Downstream processing

DOWNSTREAM PROCESSING

“BEGINNING AFTER THE END”

PRESENTED BY: AYUSH JAIN (ALM 3008)

AT WHICH STAGE ARE WE IN THE PRODUCTION

CYCLE ??

02/05/2023Basic Science H.O.T. 2


WHAT IS DOWNSTREAM PROCESSING ???

The various process used for actual Recovery and purification of biosynthetic products from a fermentation or any other industrial process together constitute a downstream processing.


WHAT ARE THOSE PROCESSES ???

Removal of

insoluble

Isolation or extraction of product

concentration of product

Packaging of product


WHAT ARE THE CLASSES OF PRODUCT OBTAINED???

Cell itself• Bakers yeast• Bio-fertilizers

Extracellular type• Antibiotics• Alcohol

Intracellular type

• Enzymes• Vitamins


IF THE PRODUCT IS THE CELL ITSELF ???

Separation of particles• Filtration• Centrifugation• Flocculation and floatation

Cooling and packaging



IF THE PRODUCT IS EXCRETED OUTSIDE THE CELL ??

• Centrifugation or filtration• Flocculation and floatation

Separation of Particles

• Liquid-liquid extraction• Whole broth extraction• Aqueous Multiphase Extraction

Extraction

• Chromatographic techniques• Evaporation process• Crystallization• Adsorption

Concentration02/05/2023Basic Science H.O.T.

9


IF THE PRODUCT IS INTRACELLULAR ???

• Filtration• Centrifugation• Flocculation

and floatation

Separation of particles

• Mechanical cell disruption

• Drying• Lysis

Disintegration of cells • Liquid-liquid

extraction• Whole broth

extraction• Aqueous

multiphase extraction

Extraction

• Evaporation• Membrane

filtration• Chromatography

techniques• Adsorption

Concentration


LETS UNDERSTAND VARIOUS TECNIQUNIES INVOLVED IN DOWNSTREAM PROCESSING

1. Filtrationfilamentous fungi filamentous bacteria

Yeast

Filtration Types • Surface filtration• Depth• Centrifugal• Cross flow• Rotatory drum

vacuum02/05/2023Basic Science H.O.T. 12


Surface filters

Filtration on the basis of pore size of

the membrane.

Cross flow filters

Filtration on the basis of

pore size but pores are not clogged due to tangential

flow of particles

Rotatory flow filters

Filtration due to sucking of

liquid by vacuum

when filter drum is

rotated in the slurry.

Depth filtersUses a porous

filtration medium to

retain particles

through out the medium rather than

just on surface

Centrifugal filters

Filtration by rapidly rotating

slurry, so solids are

retained on porous screen

and liquid filter out


2. CENTRIFUGATION

Works on sedimentation

principle

The centripetal acceleration

causes denser substances and

particles to move outward and less

dense are displaced and move to the

centre


3. FLOCCULATION AND FLOATATION

Flocculation is the process by which individual

particles aggregate to form a clump

Due to addition of clarifying agent

Floatation is the process of separation of particles based on

relative buoyancy of particlesFroth flotation

Dissolve air flotationInduced gas flotation


4. CELL DISRUPTION

Ultra sound Bead mill Homogenisers

Drying

Lysis by osmotic shock

Frezzing

Lytic enzymes

Mechanical cell lysis Non-mechanical cell lysis



5. EXTRACTION OF PRODUCT

Liquid-liquid extraction

Separation of compound based on

there relative solubility's in two

different immiscible liquids

Whole brothBoth cells and broth

are extracted together

Aqueous multiphase extraction

Enzymes are extracted in aqueous polyethylene glycol-

dextran mixture, which form separate

phases


6. CONCENTRATION

Evaporation Membrane filtration

Ion exchange

chromatography

Adsorption resins


7. PURIFICATION

CrystallizationImpure substances are

dissolved in solvent to reach saturated stage at higher temperature which is then

cooled to form pure crystalsAntibiotics like penicillin G,

Citric acid, Sodium glutamate

Chromatographic

techniques


Paper chromatographyThin layer chromatography

Column chromatographyIon exchange chromatography

Affinity chromatographyHigh performance liquid

chromatographyReversed phase high performance liquid

chromatographyGas chromatography-mass

spectrometry

Gel filtration –chromatography/ size exclusion

chromatography

Types of chromatography


PAPER CHROMATOGRAPHY

Stationary phase

Solid (paper)

Filter PaperFilter Towel

Mobile phase

Organic solvent

Water, rubbing alcohol

Characteristics

Based on

polarityAnd Size

Amino Acids

Mechanism

If molecule to be

separated are more

attracted to stationary

phase then it will move slowly and

vise versa.

Advantage

1.Cheaper2. Both

organic and inorganic

compounds can be

identified

Disadvantage

1. Large quantity of

sample cannot be applied

2.complex mixture

cannot be separated3. Less accurate



THIN LAYER CHROMATOGRAPHY

Stationary phase

Solid

Glass slide with silica

gel, Aluminum

oxide

Mobile phase

Organic solvent

Ethanol, water, hexane, pentane

Characteristics

Attraction of

compound to solid or

liquid phase

Mechanism- Based on retention factor Rf

- Higher the Rf higher the rate

with which product has

travelled

Advantages

- Simple- Cheap- Easy

recovery of product

Disadvantages

- Plate length limited so

separation up to limited

length- Sample

affected by Humidity and Temperature



COLUMN CHROMATOGRAPHY

Stationaryphase

Silica gel

Alumina

Mobile phase

Solvent accordin

g to stationary phase

Organic solvents

Characteristics

Differential

adsorption of

substance by

adsorbent or

relative affinity

MechanismBased on retention factor Rf

- Higher the Rf higher the rate

with which product has

travelled

Advantages

- Low cost-

Disposability of stationary phase so less

cross contaminatio

n

Disadvantages

-Time consuming

- Low separation

power-

Irreproducible result



GAS CHROMATOGRAPHY-MS

Stationary phase

Volatile liquid

Mobile phase

Gas(carrier)

Inert gas(He,

Ar, Ne)

Characteristics

volatility

Polarity

MechanismSeparation

due to equilibrium established between the solutes and stationary

phase

Advantages-Fast

-Sensitive-High quality product obtained-More

resolution

Disadvantages

-Apply to only volatile

compound-Performed at

higher temperature so thermally

liable compounds cannot be analyzed

-Detectors used are

destructive


Retention timeTime taken by compound

to be detected

Nature of stationary phase

Solubility of gas in solid phase

Length of column

Temperature of columnFlow rate of mobile

phase



MASS SPECTROMETRY








ADVANTAGES AND DISADVANTAGES OF HPLC

ADVANTAGES

• Can be applied to any compound soluble in liquid phase ex. AA, Lipids, Carbohydrates

• Performed at room temperature so thermally compounds can analyzed

• More flexible • detectors can be reused i.e. they are non destructive

Disadvantages• Slower technique than GC• Lower resolution when compared to GC.


ION-EXCHANGE CHROMATOGRAPHY

Stationary phase

Solid

Cellulose,

Agarose

Mobile phase

liquid

characteristics

polarity

Solubility

MechanismCharged

molecules attaches to the oppositely charged stationary phase as a result

the molecules are separated and the

molecule of interest can be

eluted by adding another compound

which has more affinity for those

charged sites

AdvantagesMore matrix

tolerance Sensitive

High quality product

obtained

Disadvantages

Expensive less

Reproducible




GEL- FILTERATION/ MOLECULAR EXCLUSION CHROMATOGRAPHY

Stationary phase

Beads with

holes

hydrocarbon like dextran

Mobile phase

LiquidOrganic solvent

characteristics

Size

MechanismBased on size proteins can pass through the holes in beads as a result pass the column

first and vice versa

AdvantagesGood

separation Biological activity of particles protectedLess and

defined timeNo sample

loss

Disadvantages

Molecules with exact same size

and weight cannot be separated



SDS- PAGE

Proteins are purified

setup similar to gel

electrophoresis

Based on size

Forces applied Frictional force and

Electric force (Gravitational force can be neglected)

Velocity depends on

Electric field, Charge on

molecule and coefficient of

fricition

Polyacramide gel is

unreactive

It has many pores to allow

proteins to pass

SDS anions and Beta

mercaptoethanol denatures the

protein by attaching to side

chains.

So net charge becomes

negative on protein



8. PACKING

Drying Spray Vaccum

Freeze drying


DOWNSTREAM PROCESS DEVELOPMENT IN BIOTECHNOLOGICAL ITACONIC ACID MANUFACTURING

APPLIED MICROBIOLOGY AND BIOTECHNOLOGYISSN: 0175-7598 (PRINT) 1432-0614 (ONLINE

JANUARY 2017, VOLUME 101, ISSUE 1, PP 1–12ANTONIO IRINEUDO MAGALHÃES JR.

JÚLIO CESAR DE CARVALHOJESUS DAVID CORAL MEDINA

CARLOS RICARDO SOCCOL

ABSTRACT• The Most Important Uses Of This Biomonomer Is The Environmentally Sustainable Production Of Biopolymers

• Separation Of Itaconic Acid From The Fermented Broth Has A Considerable Impact In The Total Production Cost.

• Previous Studies On The Separation Of Itaconic Acid Relying On Operations Such As Crystallization, Precipitation, Extraction, Electrodialysis, Diafiltration, Pertraction, And Adsorption.

• Despite Recent Advances In Separation And Recovery Methods, There Is Still Space For Improvement In Ia Recovery And Purification.


https://link.springer.com/journal/253/101/1/page/1


INTRODUCTION• Downstream Processes, Such As Recovery And Purification, Result In 30 To 40% Of The

Final Product Cost (Straathof 2011). A Competitive Bioprocess Is Highly Dependent On The Development Of Efficient Recovery And Low-cost Processes

• There Are Two Main Routes For The Production Of Biobased IA: The Direct Fermentation Into The Acid, Or The Production Of Citric Acid Followed By Its Conversion Into IA By Pyrolysis.

• Direct Fermentation Of Glucose Into Ia Is More Efficient Than The Chemical Process.

• The Price Of Ia Ranges Between Us$1.5 And 2.0/Kg And The Annual World Production Of IA Exceeded 80,000 T (Okabe Et Al. 2009). It Is Estimated That The World Market Of IA Was Valued At US$126.4 Million In 2014 And Will Reach US$204.6 Million By 2023

• Crystallization Is The Most Usual Unit Process For Recovery Of IA.

https://link.springer.com/article/10.1007%2Fs00253-016-7972-z#CR86


PHYSICAL AND CHEMICAL PROPERTIES OF ITACONIC ACID

• IA Is A White, Crystalline, Monounsaturated Organic Diacid With Formula C5H6O4 And A Molar Mass Of 130.1 G/Mol

• Solubility In Water Of 83.1 G/L At 20 °C

• The Melting And Boiling Points Are 167–168 And 268 °C, Respectively

• IA Has Three Different Protonation States (H2ita, Hita−, And Ita2−) With Dissociation Constants In Aqueous Solutions Of 3.83–3.89 (Pka1) And 5.41–5.46 (Pka2) At 15 To 45 °C

• Besides Its Two Carboxyl Groups, IA Has A Methylene Group. This Functional Diversity Allows A Variety Of Reactions, Such As Complexation With Metal Ions, Esterification With Alcohols, Production Of Anhydrides And Polymerization By Addition And By Condensation

• The Solubility Of IA Is Highly Dependent On Temperature


RECOVERY METHODS• After Conversion Of Substrates Into Itaconic Acid By Fermentation, The Resulting Broth Contains Also

Biomass, Residues From The Fermentation Media, Other Organic Acids, And Minor Components

• The Initial Step In The Downstream Process Usually Consists Of Biomass Removal, After Which The Acid Is Concentrated And Purified To The Desired Extent. An Initial Concentration (Recovery) Of The Acid From The Broth Is Central To IA Production: Lower Flows And High Final Concentrations Will Reduce Overall Operating And Equipment Costs

CRYSTALLIZATION• The Classical Method Of IA Recovery Produced By Fermentation Processes Is Crystallization.

• Ia Can Be Easily Recovered Through This Method By Cooling Or Evaporation-crystallization At Low Ph Values, But Both Treatments Do Not Separate Some By-products Of Fermentation, Such As Succinic, Malic, And Α-ketoglutaric Acid, Causing A Decrease In The Final Product Quality.

• The Filtrate Is Concentrated By Evaporation To Achieve A Concentration Of 350 G/L, And The Crude Crystals Are Formed Using A Cooling Crystallizer At 15 °C.


PRECIPITATION• IA Can Be Recovered By Precipitation With Calcium And Lead Salts (Kobayashi And Nakamura 1971)

• This Precipitate Is Filtered And Then Separated From The Liquid Phase.

• The Lead Itaconate Can Be Regenerated By Adding Carbonate Or Bicarbonate Of Alkali Metals Or Ammonium To Obtain The Respective Itaconate Salts And Lead Carbonate.

• To Isolate The Ia, It Is Necessary To Use A Cation Exchange Step

• Lead Solutions Require Appropriate Chemical Processing Due To Its High Toxicity, Which May Cause The Increase Of The Final Cost Of The Product.

• Another Ia Precipitation Method Uses Calcium Hydroxide. This Method Is Similar To The Recovery Of Other Organic Acids, Such As Citric Acid

• In This Method, Calcium Itaconate Is Formed, And Since It Is Much Less Soluble Than The Acid, It Precipitates. The Solid Is Recovered By Filtration. The IA Can Be Regenerated By Reacting Calcium Itaconate With Sulfuric Acid

02/05/2023Basic Science H.O.T.

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LIQUID-LIQUID EXTRACTION• The Use Of Conventional Solvents, Such As Long-chain Alcohols, Esters, And Alkanes Is Not Effective For

The Recovery Of Organic Acids Due To The Low Distribution Coefficient Of The Acid, I.E., Its Higher Solubility In Water Than In Organic Solvents

• The Distribution Coefficient May Be Altered Using Organophosphorus Compounds, Or Tertiary Or Quaternary Amines. This Technique Is Called Reactive Extraction (RE).

• Organophosphates And Aliphatic Amines Have Been Studied As Extractants For The Separation Of Ia From Aqueous Phase Due To Their Thermal Stability And Their Ease Of Regeneration, Which Can Be Done By Simple Distillation

• Tri-n-octylamine (TOA), A Long-chain Aliphatic Amine, Is More Effective In The RE Processes Of IA Than Tri-n-butylphosphate (TPB), An Organophosphate, Using Hexane As Diluent.

• Re System Has Numerous Advantages Such As High Productivity, Increased Conversion Of Substrate To Product, Assists In Maintaining Ph Without The Addition Of Basic Solutions, And Can Make The Continuous Process And More Energy Efficient



MEMBRANE SEPARATION• Diafiltration Is A Method Of Separation Or Removal Of Components Present In A Solution By Means Of

Permeable Membranes And A Concentration Gradient

• This Separation Process May Use Incorporated Membranes In Bioreactors For The Continuous In Situ Product Recovery (ISPR).

• Carstensen Et Al. (2012) Developed A ISPR With Diafiltration Concept Called “Reverse-flow Diafiltration” (RFD). The RFD Process Yields A Product Stream Through A Hydrophilic Ultrafiltration Hollow-fiber Membrane Immersed In The Bioreactor

• The Pertraction Technique Uses An Organic Solvent To Extract The Solute From The Aqueous Phase By A Procedure Similar To Liquid-liquid Extraction; However, The Solvents Are Separated By A Hydrophobic Membrane

• Membrane Processes Have A Relatively High Capital Cost, Can Be Selective, And With The Increasing Options Of Membrane Modules On The Market, Is A Real Possibility For Itaconic Acid Downstream.


ADSORPTION• Ia Can Be Desorbed From The Resin, Which Can Be Reused Multiple Times. There Is A Wide Range Of

Adsorbents For Adsorption Processes, Such As Alumina, Activated Carbon, Silica, And Several Kinds Of Synthetic Ion Exchange Resins.

• Gulicovski Et Al. (2008) Found That The IA Adsorption On The Surface Of Alumina Is Extremely Ph Dependent, And The Maximum Adsorption Occurred At A Ph Value Of The First Dissociation Constant, Pka1

• Separation Of IA By Adsorption From Aqueous Solutions, Using Two Types Of Commercial, Strongly Basic Ion-exchange Resins: Purolite A-500P And PFA-300. The Resin PFA-300 Proved To Be More Efficient For IA Recovery.

• In The Desorption Step, An Adequate Solution, The Eluent, Is Used To Induce The Desorption Of Ia And Carry It Out Of The Column. The Nature Of The Eluent Depends On The Type Of Adsorption:

• Adsorption Has A Relatively High Capital Cost For The Production Of Bulk Chemicals, Because Of The Cost Of The Adsorbent.




FINAL REMARKS AND FUTURE OUTLOOK

• The Development Of An Efficient Process For Separating And Purifying Itaconic Is Challenging Due To The High Affinity Of This Hydrophilic Solute For Aqueous Solutions And The Complex Composition Of The Fermentation Broth. The Advantages And Weaknesses Of Each Separation Method For IA Can Be Compared Using Literature Yields And Regarding Technology Maturity, Scalability, Costs, Energy Consumption, By-product Formation, Yield, And Purity. Conceptual Separation Trains Relying On Each Recovery Technology Were Evaluated And Summarized In Fig. All Processes Start With The Fermentation Broth And End With IA Crystals, The Preferred Product Presentation, But With Different Yields And Purity According To The Recovery Options.

• The Industrial Production Of Cheap, High-quality Itaconic Acid Depends On The Successful Recovery And Purification Of Itaconic Acid From Fermentation Broths With Low Demand Of Energy And Reagents. From The Analysis Of The Operations, It Is Clear That Further Research Laboratory To Pilot-scale Techno-economic Analysis And Environmental Impact Of Each Recovery Option Is Paramount




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