purification tutorial 2004

7/28/2019 Purification Tutorial 2004

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Introduction to Purification

Many biological processes require a purification scheme to

reduce the fermentation broth to its pure final product. Once

citric acid is made in the production fermenter, the broth is still

highly contaminated. There are many different types of

purification methods (extraction, filtration, coagulation, etc.) butfor citric acid recovery, the most commonly used method is

precipitation with lime and sulfuric acid and filtration to produce

free citric acid.

The following tutoring will go through various purification

techniques and then take you through a typical purification

scheme for citric acid.


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Types of Purification Efforts

A purification scheme can be made up any

combination of the following isolation efforts

Extraction

Precipitation

Coagulation and Flocculation

Centrifugation

Filtration

Wang 238


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Extraction

Extraction is used to liberate a product of microbial growth fromthe cells or cellular constituents that served as the enzyme sourceeither by mechanical or non-mechanical means.

Mechanical Extraction

Mechanical disruption of the cell is easy to achieve on asmall scale but can fail when used industrially

High Pressure Homogenizer- A positive displacement pump with an adjustable valve, has been

used to break microorganisms likeAspergillus niger,Escherichia

coli, andBacillus megatherium.

- When cell concentration is high, the spores or mycelia from themicroorganism can clog the valve

High Speed Ball Mill- Used for release of proteins within yeast Wang 241


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Extraction

Non-mechanical Extraction Desiccation

- Air drying that must be followed by buffer extraction

Physical and Chemical Lysis

- Osmotic shock produced by an abrupt change in saltconcentration of the medium

Solvent Extraction

- Liquid extraction of a product from soluble particles within the cell

- Must choose solvent accordingly, and purification efforts willfollow to recover product from solvent

Wang 247


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Precipitation

Precipitation is a procedure where the addition of a ionic solutionto an ionic fermentation broth forms insoluble particles, wherethe desired product is usually contained within those particles.Ionic fermentation broths usually consist of enzymes or proteins.The ways to precipitate out a product can vary from simple pH

and temperature changes to chemical reactions involving metalions. Precipitation reactions are carried out in reactors,continuous and batch.

Temperature and pH variation

Overall, most proteins and enzymes display increased solubilitywith increased temperature, but care must be taken to prevent lossof product

By adjusting the pH, an enzymes polarity can be lowered so that ithas a zero net charge; at this lowest polarity, the enzyme has low

solubility in an aqueous solutionWang 250


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Precipitation

Precipitation by Organic Solvents By adding an organic solvent to an aqueous fermentation broth, the

dielectric constant will decrease causing the solubility to decrease

Often used industrially because its inexpensive and simple

Precipitation by Metal Ions Metal salts with lower solubilities can formed by enzymes and

proteins

Nucleic acids, which are present in microbial cells, must beremoved prior to this type of precipitation because they reduce the

resolution of separation Manganous salts can be used to selectively precipitate out those

nucleic acids

Wang 255


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Coagulation is defined for biological processes to be when smallparticles directly adhere to each other, while flocculation is when

an agent acts as a bridge that joins particles together.

Coagulation and flocculation techniques are usually applied to

either whole cells, cell debris, or soluble proteins.

Whole Cells Many flocculation agents are used to separate products, such as

anionic and cationic ployelectrolytes, polyamines, alumina, andsynthetic polymers

Less information is known about coagulants, but some studiedinorganic coagulants have been alum, ferric salts, and calcium salts

Wang 256


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Cell Debris and Proteins Coagulation and flocculation are useful techniques in removing the

cell debris that can be produced during mechanical agitation

Coagulation and flocculation can be used alternatively to

precipitation methods to remove enzymes

The same agents for whole cell removal can be applied to cell

debris and protein removal

Wang 256


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Centrifugation

Centrifugation involves separation of liquids and particlesbased on density. Centrifugation can be used to separate cellsfrom a culture liquid, cell debris from a broth, and a group of

precipitates. There are numerous types of centrifuges, but only afew will be presented here.

Tubular Bowl Centrifuge Most useful for solid-liquid separation with enzymatic isolation

Can achieve excellent separation of microbial cells and animal, plant, andmost microbial cell debris in solution

Disc Bowl Centrifuge Widely used for removing cells and animal debris

Can partially recover microbial cell debris and protein precipitates

Wang 261


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Centrifugation

Perforate Bowl Basket Centrifuge Exception at separation of adsorbents, such as cellulose and agarose

Zonal Ultracentrifuge

Applied in the vaccine industry because it can easily remove celldebris from viruses

Can collect fine protein precipitates

Has been used experimentally to purify RNA polymerase and very

fine debris in enzymes

Wang 261


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Filtration

Filters use a filter cloth or some porous material along withapplied pressure to push smaller particles through the filter, thusseparating elements of the solution based on size. Filtration for

biological materials is generally completed using batch filtration,rotary drum filtration, or ultrafiltration methods.

Batch Filtration Usually performed under constant pressure with a pump that moves

the broth or liquor through the filter

Filter cake will build-up as filtration proceeds and resistance to

broth flow will increase The filter press is the typical industrial version of a batch vacuum

filter, using a plate and frame arrangement

Can be used to remove cells, but does not work particularly well foranimal cell debris or plant seed debris

Wang 267


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Filtration

Rotary Drum Filtration Solution is vacuumed upward where it crosses a filter septum

removed by a positive displacement pump

Filter cake is removed after each rotation to give a fresh surface forfiltration

Rotary vacuum filters can be used to efficiently remove mycelia,cells, proteins, and enzymes, though a filter aid or precoat of theseptum may be necessary

Ultrafiltration

Utilizes a membrane to separate particles that are much larger thanthe solvent used

Successful removal occurs in the partical size range of 10 solventmolecular diameters to 0.5

Wang 270


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Purification of Citric AcidA typical method used for purification of citric acid from a

fermentation broth involves two major purification techniques:

precipitation and filtration. The following schematic displays a

generic citric acid purification scheme. The scheme will be

discussed in detail in the next few slides.

ffffPrecReact citric acid

with calcium

carbonate

Filter

precipitateReact precipitate

with sulfuric

acid

Filter

precipitatePurifiedCitric

Acid


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Purification of Citric AcidThe citric acid broth from the production fermenter is highly

contaminated by leftover biomass, salts, sucrose, and water. First,

the citric acid must be reacted with calcium carbonate to

neutralize the broth and form the insoluble precipitate calcium

citrate. Calcium citrate contains about 74% citric acid. The

stoichiometric equation is as follows:

CaCO3+ Citric Acid CO2+ Calcium Citrate

Contaminated Citric Acid

CSTRCalcium Citrate as

a precipitate plus

contaminants

Calcium Carbonate, CaCO3


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Purification of Citric AcidThe calcium citrate is then washed, heated, and filtered to remove

any number of the contaminants. Depending on the specific

design of the purification scheme, filters can be placed before the

first reaction with calcium carbonate, in series between the two

precipitation reactions or in any other combination that works.

Also, it is important to choose the best kind of filter for what isbeing removed. For simplicity, the filters here will remove larger

contaminants first (sucrose and salts) and the smaller

contaminants later.

Kirk 15

Calcium Citrate as

a precipitate plus

contaminants

Filter

Ex. Plate filter, Rotary

presses, rack-and-

frame presses

Calcium Citrate,

biomass, water


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Calcium Citrate,

biomass, water

Purification of Citric AcidTo crack the calcium citrate precipitate, sulfuric acid is needed.

The temperature of this reaction should stay below 60C. Thereaction will produce free citric acid and a new precipitate,calcium sulfate, which will need to be removed later. Thestoichiometric coefficients for this reaction are all one.

Kirk, 15

CSTR Calcium sulfate as aprecipitate, free citric

acid, biomass, water

Sulfuric

Acid, SO4


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Purification of Citric AcidIn this filter, the calcium sulfate is washed away from the citric

acid and the leftover biomass is removed. Again, thecontaminants that were present in the fermentation broth can beremoved by additional filtration means, such as microfiltration orultrafiltration.

Kirk 15

Filter

Ex. Plate filter, Rotary

presses, rack-and-

frame presses

Citric acid,

water

Calcium sulfate as a

precipitate, free citric

acid, biomass, water


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Purification of Citric Acid

Further PurificationCitric acid can be produced in two formsmonohydrate andanhydrous. These forms may require additional purification steps

to reach the desired purity.

1. Monohydrate contains one water molecule for every citric acid

molecule

Requires repeated crystallization until water content is approx. 7.5-8.8%

2. Anhydrous Processed to remove all water from end product

Prepare by dehydrating the monohydrate citric acid product at atemperature above 36.6C

Kirk 16,17

KICgroup 1


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Purification Conclusion

Once the product has been brought to the desired purity, it wouldbe sent to packaging and distribution.

In summary, there are many different methods and types ofequipment that make up a purification scheme. Some of the

more common types or purification were discussed in the firsthalf of this tutorial. The second half of the tutorial dealt with aspecific citric acid purification scheme that utilizes precipitationand filtration to recover free citric acid from a contaminatedfermentation broth.

Citric

AcidPurification


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References

KIC Chemicals. Citric Acid Monohydrate, USP (Coarse-Granular). Retrieved April 21, 2004, from the World WideWeb: http://www.kicgroup.com/citmonokc.htm

Kirk, Raymond E., Othmer, Donald F. (Ed). (1949).

Encyclopedia of Chemical Technology (Vol 4). New YorkCity: Interscience Publishers, Inc.

Wang, Daniel I. C., & Cooney, Charles L., Demain, Arnold L.,

Dunnill, Peter, Humphrey, Arthur E, Lilly, Malcolm D.

(1979). Fermentation and Enzyme Technology. NewYork City: John Wiley & Sons.

Citric

Acid

purification tutorial 2004

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