Downstream processing

DOWNSTREAM
PROCESSING
“BEGINNING AFTER THE END”
PRESENTEDBY: AYUSHJAIN
(ALM3008)

AT WHICH STAGE ARE
WE IN THE
PRODUCTION CYCLE
??
18-03-2017Basic 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
Concentration
18-03-2017
Basic 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. Filtration
filamentous fungi
filamentous bacteria
Yeast

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 filters
Uses a
porous
filtration
medium to
retain
particles
through out
the medium
rather than
just on
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 particles
Froth flotation
Dissolve air flotation
Induced 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 broth
Both 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
•Crystallization
•Impure substances are
dissolved in solvent to reach
saturated stage at higher
temperature which is then
cooled to form pure crystals
•Antibiotics like penicillin G,
Chromatographic
techniques

Paper chromatography
Thin layer chromatography
Column chromatography
Ion exchange chromatography
Affinity chromatography
High performance liquid
chromatography
Reversed phase high
performance liquid
chromatography
Gas chromatography-mass
spectrometry
Gel filtration –
chromatography/ size exclusion
chromatography
Types of
chromatography

PAPER CHROMATOGRAPHY
Stationary
phase
Solid
(paper)
Filter
Paper
Filter
Towel
Mobile phase
Organic
solvent
Water,
rubbing
alcohol
Characteristics
Based on
polarity
And 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.Cheaper
2. Both
organic and
inorganic
compounds
can be
identified
Disadvantage
1. Large
quantity of
sample
cannot be
applied
2.complex
mixture
cannot be
separated
3. 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
Stationary
phase
Silica gel
Alumina
Mobile
phase
Solvent
according to
stationary
phase
Organic
solvents
Characteristics
Differential
adsorption
of substance
by
adsorbent or
relative
affinity
Mechanism
Based 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
Mechanism
Separation
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 time
Time taken by compound
to be detected
Nature of stationary phase
Solubility of gas in solid phase
Length of column
Temperature of column
Flow 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
Mechanism
Charged 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
Advantages
More 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
Liquid
Organic
solvent
characteristics
Size
Mechanism
Based on size
proteins can
pass through
the holes in
beads as a
result pass the
column first
and vice versa
Advantages
Good
separation
Biological
activity of
particles
protected
Less and
defined time
No sample
loss
Disadvantages
Molecules
with exact
same size and
weight cannot
be separated

SDS- PAGE
•Proteins
are purified
• setup
similar to
gel
electrophore
sis
Based
on size
Forces
applied
Frictional
force and
Electric
force
(Gravitation
al 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 BIOTECHNOLOGICALITACONIC ACID
MANUFACTURING
APPLIED MICROBIOLOGYAND BIOTECHNOLOGY
ISSN: 0175-7598 (PRINT) 1432-0614 (ONLINE
JANUARY 2017, VOLUME 101, ISSUE 1, PP 1–12
ANTONIO IRINEUDO MAGALHÃES JR.
JÚLIO CESAR DE CARVALHO
JESUS 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.

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. 18-03-2017Basic Science H.O.T. 49

PHYSICALAND 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/LAt 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
18-03-2017
Basic Science H.O.T.
52

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 IAAdsorption 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-500PAnd 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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