Bioseparation Engineering

1
Bioseparation Engineering
2
Chapter 1 Introduction
3
1.1 Downstream processing in biotechnology
4

• The producing process of a biotechnological
  product is termed bioprocess, that can be divided
  into two processes as follows
• Upstream processingstrain development and
  bioreaction (enzyme catalyze reaction, microbial
  fermentation, plant/mammalian cell culture, etc)
• Downstream processingthe isolation and
  purification of biotechnological products
• The complexity of downstream processing is
  determined by the required purity of the product,
  in turn determined by its application.

5

• The downstream processing scheme normally can be
  divided into the following stages

Extracellular product
Solid-liquid separation
?
Broth
Cell disruption
Removal of cell debris
Intracellular product
?
?
Final product
Primary isolation
?
?
?
purification
formulation
?
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1.2 Demands on the downstream
processing
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• Strictly monitor the DSP steps for keeping the
  activity of the product
• Rapidly remove those impurities can affecting the
  stability of the final product
• Generally it is necessary to apply special
  efficient methods to the purification of the
  product
• Since many products are applied to food,
  pharmaceutical, and cosmetics, those substances
  harmful to mankind health must be removed
• Since the product concentration is low in the
  culture broth, it is necessary to concentrate the
  broth for removing large amounts of water.
  Because those of unit operations are costly, the
  cost of DSP is increased observably.

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1.3 Separation mechanism and unit
operation
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• According to different separation principles the
  unit operations can be sorted into two groups
• Mechanical separation
• Objectunhomogeneous phase system
• Mechanismseparation based on the difference of
  substances size and density
• Unit operationfiltration, settling,
  centrifugation, etc.
• Mass transfer separation
• Objecthomogeneous phase system
• Transport/velocity separation
• Mechanismseparation based on the migration
  velocity difference of solutes drove by bearing a
  applied force
• Unit operationultrafiltration, reverse osmosis,
  electrophoresis, etc.
• Diffusion/equilibrium separation
• Mechanismseparation based on the difference of
  distribution of substances between the two phases
• Unit operationextraction, crystallization,
  adsorption, ion exchange, etc.

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1.4 Estimation for separation
efficiency
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• There are three criteria on assessing the
  efficiency of a downstream processing, i.e.
  concentration degree, isolation-purification
  degree, and recovery rate
• Concentration degree
• Generally can be represented as concentrated
  factor

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• Isolation-purification degree
• Can be represented by separation
  factor/coefficient

• Recovery rate

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Chapter 2 Solid-liquid separation and
cell disruption
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2.1 Cell separation
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2.1.1 Settling

• Stokes settling velocity of global particle

Where d is particle diameter, ?s and ?L are the
density of particle and liquid, separately, ? is
resistance coefficient, Re is Reynolds number,
and ?L is the viscosity of liquid.
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• The volume of the cells is so small that its
  settling velocity is very slow. For accelerating
  settling process agglomeration of individual
  cells into large flocs is done by the addition of
  flocculating agents such as polycations, or
  inorganic salts.

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2.1.2 Centrifugation

• Centrifugation velocity

Where r is centrifugal radius, ? is rotary
angular speed, N is revolution of centrifuge, and
S is sedimentation coefficient.
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• Centrifugation
• Differential centrifugation
• It is an unit operation commonly used in the
  biochemical industry. According to the
  characteristics of the broth, the aim of
  isolation, and the extent of separation
  requested, different components can be separated
  from the broth separately by selecting proper
  operational parameters in practice.
• Zonal centrifugation
• Rate-zonal density gradient sedimentation
• Isopycnic density gradient sedimentation
• Besides sucrose, CsCl and NaBr can be used for
  achieving the density gradient, and applied to
  the separation of nucleic acid and lipoprotein,
  respectively.

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• Centrifuges
• The tubular bowl rotor centrifuge is commonly
  applied on a laboratory scale, and the types of
  tubular bowl and disc stack are commonly used on
  an industrial scale.

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• The processing capacity of the tubular bowl
  centrifuge is described by

Where L is the length of the tube, r2 is the
inside radius of the tube, and ? is usually
called the area of centrifugal sedimentation, a
function of the structure of the centrifuge and
the operating conditions.
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2.1.3 Filtration

• Definitiona technology, apply filter media to
  retain the particle while allowing the passage of
  the liquid through the filter, is used to achieve
  solid-liquid separation.

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• The flow through the filter

Where Q is the volume of the filtrate, A is the
area of the filter, ?p is the pressure
difference, ?L is the viscosity of the filtrate,
Rm and Rc are the resistance of the filter medium
and the cake, ? is the average specific
resistance of the cake, and W is the weight of
the dried cake.
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• Before filtration pretreatment of the broth by
  addition of flocculating agents, their function
  have been described in Section 2.1.1, and
  precoating the filter medium with filter aids
  (diatomite, perlite, etc.) are usually required
  to improve the filtration velocity.
• Filtration equipment
• Filter press and rotary drum vacuum filter are
  frequently used for clarification of the broths.

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2.2 Cell disruption
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• Many biotechnological products cant be excreted
  outside of the cells during microorganism grow.
  For collecting those products the first step
  must be rupturing of the microbial cells to
  release the intracellular compounds into the
  liquid phase.

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2.2.1 Cell structures

• The cell structures are quite different among a
  considerable variety of cells. The sequence of
  different cells being broken from difficult to
  easy can be listed as follows
• plant cells, yeast cell, G cells, G- cells,
  and animal cells.
• The goal of cell disruption is making the cell
  wall and/or cytoplasmic membrane damaged more or
  less to liberate the intracellular products .

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2.2.2 The principles of cell disruption

• Mechanical disruption
• The Cells structure is broken due to the cells
  being sheared and pressed by mechanical forces.
  As a general rule, the more small the size of the
  cells is, the more hard to be ruptured it is.
• Chemical/enzymatic means
• Treatment with chemicals/enzymes can damage the
  cell membranes/walls and render cells more
  permeable, that is available for release of
  intracellular products.

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2.2.3 The means of cell disruption
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• Mechanical disruption
• High-pressure homogenisation
• Principle the cell suspension is forced at high
  pressure through an orifice of narrow internal
  diameter to emerge at atmospheric pressure. The
  sudden release of pressure creates a liquid shear
  capable of disrupting the cells.

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• The influencing factorspressure, cyclic times,
  temperature, etc.

Where S is the disruption scale, p is operational
pressure, N is cyclic times, k is the disruption
velocity constant, correlation with the kind of
the cells and operational temperature.

• CharacteristicIt is feasible for disruption of
  yeast cells and the majority of bacteria cells,
  but not suitable for disruption of filamentous
  fungus.

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• Bead milling
• Principleagitation with glass in bead mills
  ruptures the cells by a combination of high shear
  and impact with the cells.

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• The influencing factorsagitation speed, the
  concentration of cells, the operating time, the
  beads diameter, density, and loading density.

Where S is disruption ratio,k is disruption
velocity constant, correlation with the beads
diameter, density, loading density, the
concentration of cells, agitation speed, and the
shape of the puddler, t is the operating time of
batch operation, or can be written as tV/Q (V is
the effective volume of the chamber of the bead
mill, and Q is the feed flux) at continue
operation.

• Characteristic The method can be widely
  applied to a variety of cells, but it is very
  poor on the available energy, the ability of the
  heat change must be considered in the cooling
  system design. And because many operating
  parameters can influence the disruption ratio,
  optimizing design of the processing is very
  complex.

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• Ultrasonication
• Principlecavitation.
• The influencing factorsthe kind and
  concentration of the cells, and the energy of the
  ultrasonication.
• Characteristic it is commonly used at laboratory
  scale removal of the heat generated is difficult
  on a larger scale.

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• Chemical methods
• Treatment with chemicals
• Principlesee 2.2.2
• Available chemicals acid, alkali, organic
  solvents, detergent, chelates, chaotropic agents,
  etc.
• Enzymatic lysis
• Principlesee 2.2.2
• Available enzymes Because there are different
  chemical components of cell wall among a variety
  of organisms, proper enzyme must be selected,
  e.g. lysozyme is suitable for treatment of
  bacteria Zymolyase, ?-1,6-dextranase, or
  mannanase is used for yeast and damaging plant
  cells need to apply cellulase.

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• A combination of enzymatic/chemical lysis with
  mechanical disintegration has been suggested in
  enhancing the efficiencies of the respective
  methods, with savings in time and energy and the
  facilitation of subsequent processing.

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• Physical means
• Osmotic shock
• Principleput cells into a solution of lower
  osmotic pressure suddenly from that of higher
  osmotic pressure, that result in a lot of water
  swarming into cells and bursting the periplasmic
  membrane.
• Characteristicit is the most mild method of cell
  disruption, but only effective for animal cells
  that lack a cell wall.

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• Freeze-thaw
• Principlebecause of water crystallizing
  quantities of crystal nucleus are formed in the
  cells during the cells are frozen rapidly, that
  can damage the structure of the cells. Generally
  freeze and thaw must be carried out again and
  again until the expectation for the ratio of cell
  disruption is met.
• Characteristicit is only suitable for those
  cells whose wall is thinner, and difficult to be
  used on a larger scale.

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• Summary Since the structure among many species
  of cell and the property of products are much
  different, choice of the disruption methods has
  to be made empirically, at the same time taking
  into consideration the subsequent processing
  steps.

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Chapter 3 Precipitation
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• Definitiona phenomena of solid aggregates formed
  in a solution, that is based on a decrease in
  solubility induced by external factors.
• Characteristic precipitation is a elementary
  isolation technique. The purity of sediment is
  much lower than that of crystal. But high-purity
  products can be gotten by multistep operation.
• Application it is widely applied to recovery of
  biotechnological products e.g. proteins.

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• Commonly used methods

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3.1 Salting-out precipitation
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3.1.1 Theory

• Definitionin a solution of increasing ionic
  strength the precipitation of proteins will
  happen, that is relative to a decrease
  solubility.
• Cohn empirical formula

Where S is the solubility of the protein, ? is a
constant, Ks is salting-out constant, I is the
ionic strength, ciand Zi are is molar
concentration and number of charge, respectively.

• Mechanism the addition of neutral salt can
  increase hydrophobic interactions between neutral
  protein molecules, that is widely accepted.

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3.1.2 The influencing factors

• The molecular weight and three-dimensional
  structure of different proteins
• for given protein
• the kind of inorganic salts (to Ks)
• Criteria of selecting a neutral salt
• higher solubility
• solubility is almost influenced by temperature
• the density of the solution in which the neutral
  salt is dissolved is not much higher, that will
  facilitate the separation of the sediments by
  centrifugation.
• Most used neutral salt is (NH4)2SO4, besides
  Na2SO4 and NaCl can also be used .

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• temperature and pH(to ?)
• under a higher ionic strength the solubility of
  proteins will decrease accompanied with the
  increase of temperature.

• when pH is close to pI,solubility of the protein
  is lowest.

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3.1.3 Unit operation of salting-out

• The experimental steps for deciding the
  saturation used to precipitate given protein
• take a small portion of material liquor, and
  equally divide into several part. And refrigerate
  to 0?
• separately calculate the additive amounts, that
  can make the solution reach the saturation from
  20 to 100 , and add according to the calculated
  results. At the same time keep the temperature at
  0?

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Where W is the additive amount(g/L), S is the
saturation, 505 is the saturated concentration of
(NH4)2SO4 at 0? (g/L), and 0.285 is the saturated
concentration of (NH4)2SO4 at 0? (L/L).

• After centrifugation dissolve the sediment and
  determine the concentration of total protein and
  that of given protein, respectively, at the same
  time determine corresponding concentration in
  the mother liquor. Compare the results and assure
  that the mensuration is reliable

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• plot a figure to describe the correlation
  between the concentration of total protein and
  that of given protein and saturation of (NH4)2SO4
  in the mother liquor,by that decide the additive
  amount based on the request for recovery of
  products.

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3.1.4 Application

• Because of many salts being remained in the
  sediment removal of salts must be carried out
  after salting-out precipitation.

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3.2 Isoelectric precipitation
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• Definitionisoelectric precipitation can be used
  for recovery of proteins, that is based on the
  principle of a decrease in solubility when pH
  of the solution is adjusted to pI.
• Operating condition
• lower ionic strength
• pH?pI

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• As a rule it is applied to precipitation of
  hydrophobic proteins e.g. casein, and not
  suitable for hydrophilic proteins e.g. glutin.
  So applying fields is not wider than salting-out
  precipitaion.
• Characteristic
• advantageit is facilitated to subsequent
  operation
• disadvantagesometimes extremes of pH denature
  the products.

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3.3 Organic solvent precipitation
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• Principlereduced dielectric constant enhances
  electrostatic interactions between protein
  molecules.
• Operating condition
• low ionic strength
• pH is near pI
• Characteristic
• advantagethe lower density of the organic
  solvent is convenient for separating sediment,
  and removal of salts isnt needed.
• disadvantageproteins denaturing maybe happen, so
  low temperature required for operation.

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3.4 Another methods
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3.4.1 Thermal precipitation

• Principleunder higher temperature make heat
  sensitive proteins precipitate and achieve the
  separation of heat stable proteins.
• Operating condition
• adjust pH of the solution
• add organic solvents.
• Characteristic
• it is a separation method of making proteins
  denaturation, so there should be a difference of
  heat stability between the given protein and the
  impurity proteins, that must be known very
  clearly.

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3.4.2 Special agents

• Non-ionic polymer
• Mechanismreduction in the effective quantity of
  water available for protein solvation.
• Agent in common usePEG
• Charged polymer
• Mechanismcomplex formation between oppositely
  charged molecules leads to charge neutralization
  and precipitation.
• Available agentsacidic polysaccharides, CMC,
  etc.

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• Polyvalent metal ions
• Mechanismbond with some functional groups in the
  surface of protein molecule e.g. Ca2 and Mg2
  can combine with carboxyl, Mn2 and Zn2 with
  nitrogenous compound and heterocyclic compound.
• Advantage
• although lower protein concentration in the
  solution precipitation can be achieved too.
• after precipitation removal of metal ions is easy
  by using ion-exchange resin or chelating reagent.

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Chapter 7 Affinity purification
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• Bioaffinity
• To carry out life functions, biological systems
  undergo physical and chemical interactions that
  rely on variations in molecular selectively and
  binding strength. The particular set of physical
  and chemical interactions in which structure can
  play a major role in shaping is referred to as
  bioaffinity.
• Affinity purification
• Bioaffinity interactions have been employed in
  bioseparation processes, that lead to the
  appearance of affinity purification techniques
  such as affinity chromatography, affinity
  membrane, affinity precipitation, etc.

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7.1 Basic principle
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7.1.1 Molecular recognition processes

• From a molecular perspective, the binding process
  between a receptor and its ligand can be viewed
  as four continuous steps
• Electrostatic interaction
• Solvent displacement
• Steric selecion
• Charge and conformation rearrangement

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7.1.2 Receptor-ligand interactions

• In the affinity, receptor-ligand interactions
  involve noncovalent interactions such as
• Ionic bonds
• Hydrogen bonds
• Hydrophobic interactions
• Van der waals forces
• Specific interactions result during the formation
  of different receptor-ligand complexes, and some
  of those can be applied to bioseparation for
  example
• Antibody-antigen interaction
• Enzyme-substrate interaction
• Lectin-carbohydrate interaction

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7.2 Affinity chromatography
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7.2.1 Principle and operation

• Principle
• Molecular recognition forms the basis of
  adsorption and separation by affinity
  chromatography. One of the reactants in an
  affinity pair, the ligand, is immobilized on a
  solid matrix and is used to fish out the aim
  product (receptor).

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• Operation
• Process including four steps, that is adsorption,
  wash, elution, and reuse.
• Elution methods
• Specific elution
• A free ligand molecule added into elution liquid.
• Nonspecific elution
• Increasing the ionic strength
• Changing the pH of the buffer

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7.2.2 ligand

• Examples of ligands used for affinity
  chromatography of protein

Ligand type Ligand type Protein type
Biospecific ligands Receptor Hormone
Biospecific ligands Antibody Antigen
Biospecific ligands Substrate/substrate analogue, inhibitor, cofactor enzyme
Biospecific ligands Lectins Glycoproteins
Pseudobiospecific ligands Triazine dyes Dehydrogenases, kinases, and other proteins
Pseudobiospecific ligands Metal ions Metal ion binding proteins
Pseudobiospecific ligands Hydrophobic groups Various proteins
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• The chemical coupling procedure for
  immobilization of a ligand is chosen so as to
  provide
• Satisfactory yields
• Strong linkage to minimize ligand leakage during
  chromatographic operation
• Minimal nonspecific interactions with
  biomolecules.

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7.2.3 Application and advantage

• A trend in downstream processing has been to
  exploit the specificity of affinity interactions
  earlier in the separation train so as to reduce
  the number of purification steps.
• Potentially, affinity chromatography possess very
  high resolving power.

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7.3 Affinity membrane
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7.3.1 Principle and operation

• Micro/macroporous membrane matrices with affinity
  ligands have been developed for binding proteins
  from the clarified feed pumped over them.
• Desorption of the protein is later performed
  using solutions as in affinity chromatography.

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7.3.2 Advantage

• Can give similar high resolution separation as
  chromatographic methods
• Increase the speed of separation tremendously due
  to in membranes, liquid transport is by
  convection as opposed to the diffusional flow in
  gels.

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7.4 Affinity precipitation
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7.4.1 Principle

• Selectivity in precipitation has been introduced
  by use of affinity interactions. Creation of
  large complexes as a result of affinity
  interactions, as between antigen and antibody, is
  one mode of affinity precipitation, which is used
  in immunoprecipitation.

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7.4.2 Application

• Selective precipitation of multimeric proteins
  (have more than one binding site for a ligand).
• The homobifunctional ligands (synthesized by
  coupling two ligand molecules by a spacer) are
  able to bridge different protein molecules
  thereby forming aggregates.
• With heterobifunctional ligands, where one
  functionality is responsible for the affinity
  binding and the other is exploited for the
  precipitation, it becomes possible to operate
  affinity precipitation in a more general mode.

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Chapter 4 Extraction
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• Principle the substances can be purified or
  concentrated since partition coefficients of them
  are different between the two phases.
• The progress of extraction technique
• traditional organic solvent extraction
• ?
• liquid membrane and reverse micelle
  extraction
• ?
• aqueous two-phase and supercritical fluid
  extraction

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4.1 Basic concepts
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4.1.1 Extraction

• definitiona unit operation employing liquid or
  SCF as solvents to extracting the product in the
  material.
• Classification

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4.1.2 Back-extraction

• Definitionan operation of target products being
  transferred from organic phase into a new aqueous
  phase under conditions different from the first
  extraction.
• Purposefor farther purifying products or
  facilitating consequent separation.

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4.1.3 Physical extraction and chemical
extraction

• Physical extraction
• principlethe compound distributes itself between
  the two phases according to its physical
  preference.
• applicationpenicillin and other antibiotics.
• Chemical extraction(reactive extraction)
• principlefat-soluble extractants can form
  fat-soluble complexes with the compound by
  chemical reaction, that carry the compound from
  the aqueous to the organic phase.
• applicationit is advantageous for compounds that
  have a high solubility in aqueous medium, e.g.
  organic acids.

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4.1.4 Distribution law

• Distribution lawunder a given temperature and
  pressure the concentration ratio of a solute
  between the two phases is a constant, that is
  called the partition coefficient m after reaching
  the distribution equilibrium.

Where CL is the concentration of substance in
extract phase, and CR is the concentration of
substance in raffinate phase.
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• Extraction factor Ethe mass ratio of target
  product between extract phase and raffinate phase
  after extraction goes to a balance.
• It is a indicator of evaluating the efficiency of
  an extraction process.
• Calculating formula

Where VL and FL are volume and flux of the
extractant, respectively, VR and FR are volume
and flux of the primary liquid, respectively.
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4.2 Organic solvent extraction
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• Characteristichigher processing capacity, lower
  power consumption, quicker speed of separation,
  and easy to achieve continue operation and
  autocontrol.
• Application extraction of small molecule
  biotechnological products e.g. antibiotic,
  organic acid , vitamin and amino acid, etc.

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4.2.1 The influencing factors

• Extractant
• desirable criterion on choosing extractant
• the analogous polarity with target product
• cheap
• water-nonmiscible
• lower density and viscosity, phase disperse and
  separation are liable
• facile recovery and recycle
• low toxicity and causticity, and safe to use.
• no reaction with the product.

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• pH in the aqueous phase
• in the case of extraction of weak electrolyte pH
  of the aqueous phase influence m (for example the
  extraction of penicillin).

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• Emulsification
• definitiona phenomena, micro-dripping of water
  (or organic solvent) diffuse into the organic
  phase (or aqueous phase), is called
  emulsification.
• resultphase separation is difficult to achieve.
• reasonproteins and other substances in the broth
  have the same function as surfactant.
• resolvent
• after emulsification apply filtration or
  centrifugation to eliminate
• best method is pretreatment of broth to remove
  proteins e.g. agglomeration.

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(No Transcript)
90
4.2.2 Operating mode and calculation

• Classification of extraction equipment

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• One-level extraction

Where E is extraction factor, and ? is mass
fraction of product between the raffinate phase
and the feed.

• Characteristicsimple, but the efficiency is low,
  the content of product is higher still in
  raffinate phase.

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• Multilevel cross-current extraction

• Characteristic
• advantagethe driving force of extraction is
  stronger, so the efficiency is higher
• disadvantagestill need add a lot of extractants,
  and the concentration of product is low, so must
  consume much energy to recover extractants.

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• Multilevel counter-current extraction

• Characteristicthe efficiency is much higher and
  the usage of extractants is less too, so be
  commonly used at the industrial scale.

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4.3 Aqueous two-phase extraction
95

• Aqueous two-phase system (ATPS)prepared by
  mixing two different polymers, or a polymer and a
  salt above certain concentrations with water as
  the major component.
• Applicationsuitable for extracting protein,
  especially intracellular protein.

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4.3.1 ATPS

• Reasonbecause of incompatibility i.e. steric
  exclusion between the molecules of different
  polymers, that make the solution trend towards
  phase separation, and form two phases on a given
  condition.
• ATPS commonly used
• polymer/polymerPEG/Dx. PEG is the main component
  in the top phase, while dextran constitutes the
  bottom phase.
• polymer/saltPEG/KPi. The top phase is enriched
  in PEG, and the bottom phase KPi.

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• Phase diagram of ATPS(binodal line)
• tie linethe straight lines connecting two dots
  on the binodal line.
• Any dots on the same tie lineconstitutions of
  the two phases are same, but volumes are
  different.

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• The length of tie linecan indicate the
  difference of property between the two phases.
  The more long a tie line is, the more different
  properties of the two phases are, specially when
  the length is equal to zero, representing the dot
  called critical point on the binodal line, there
  isnt a difference between the two phases, i.e.
  homogeneous phase reform.

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4.3.2 The distribution equilibrium in ATPS

• The partition coefficient of solute in ATPS

Where C2 and C1are the concentrations of the
solute in the top phase and the bottom phase,
respectively.

• Principal factors of influencing partition
  coefficient
• The electrostatic and hydrophobic interactions
  between solute and ATPS

Where HF and HFS are the hydrophobicities of the
ATPS and the protein, respectively, F?R and T are
faraday constant, gas constant and absolute
temperature, respectively, Z is the net charge
amount of the protein, and ?? is Donnan potential.
100
4.3.3 The factors of influencing on
extraction efficiency

• Polymer
• Molecular weightthe more light molecular weight
  of a polymer is, the more easy it is that protein
  is distributed to the phase rich in the polymer.
• Total concentrationthe more high, the more
  different the propertis of the two phases are,
  that means the tie line is longer, and protein
  more liable of distributing into one of the two
  phases.

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• The kind and concentration of salt
• Influence ??

Where m and m- are the partition coefficients of
the cation and anion of the electrolyte,
respectively, Z and Z- are the charge amounts of
the cation and anion of the electrolyte,
respectively.

• Influence ?HFSthe concentration increase of salt
  can make surface hydrophobic property of protein
  increase based on the effect of salting-out.
• Influence ATPSchange the composition of the
  phase components in the two phases, and the
  volume ratio between the two phases.

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• pH
• Influence Zthe surface charge amount of protein,
  i.e. the ionicity of protein.
• Influence ??the ionization of phosphate.
• Temperature
• Influence the phase diagram of ATPS primarily.
• Generally ATP extraction operation carries out at
  room temperature, that is because of
• the effect of PEG on the stability of protein
• the lower viscosity of solution, and being liable
  to achieve phase separation
• saving the cooling expense.

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4.3.4 ATP extraction operation

• The selection of ATPS
• According to the difference of characters e.g.
  hydrophobicity, molecular weight, isoelectric
  point, etc. between the target protein and
  impurities choosing a ATPS and adding proper kind
  and concentration of salt can achieve the
  extraction of the product.

104

• Design experiment to decide the optimal
  extraction system. Generally the test of
  distribution equilibrium carries out by using
  multigroup 10mL centrifugal tubes.
• prepare higher concentration solution of polymers
  and salts, so get a series of ATP with different
  concentration, pH, and ion strength
• after addition of material liquor dilute to 10mL,
  and mix to achieve the extraction
• centrifugate and make the two phases separate
  completely
• determine the concentrations or bioactivities of
  the target product in the top and bottom phase
  respectively, and calculate the partition
  coefficient.

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• Extraction of intracellular protein
• Advantagemake the cell debris distribute into
  the bottom phase, and the product distribute into
  the top phase, that can achieve the purifying of
  product partly, and the removal of debris at the
  same time.
• Operation in practice
• phase dispersedirectly mix solid or concentrated
  polymers and salts with homogenate, and stir to
  make it dissolve and form ATP, and reaching to
  partition equilibrium spend about several seconds
  because of the lower surface tension in the ATPS
• phase separationcan be speeded up by
  centrifugation, and phase separation can finish
  within less than 40 seconds for cell debris in
  the extraction system.

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4.3.5 Application and advantage

• Multistep extraction

107

• ATP extraction at a large scale

108

• Advantage
• high capacity (biomass to volume ratio)
• straightforward scale-up
• adapted to the extraction equipment used for
  water-organic solvent systems.

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4.4 Liquid membrane extraction
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• Definitionliquid film composed of aqueous
  solution or organic solvent, that divide
  nonmiscible liquids, and permit solute to
  permeate from one side of the membrane to the
  other side selectively.
• Characteristichigher efficiency, and specially
  can achieve extraction and back-extraction at the
  same time.
• Applicationseparation and purification of small
  molecular biotechnological products, e.g. organic
  acid, amino acid, antibiotic, etc.

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4.4.1 The kinds of liquid membrane

• Emulsion liquid membrane (ELM)

• The type of (W/O)/W is primarily applied to
  bioseparation, and its inner phase and outer
  phase are all aqueous solution, while the major
  component of membrane is organic solvent.

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• Construction and composition

• Preparing process
• Add water into the organic solvent, in which
  surfactant and additive are dissolved, then the
  emulsion of W/O is formed by high speed stirring
  or supersonic.
• Dispersing the prepared emulsion into second
  aqueous phase, i.e. emulsifying secondly, can get
  (W/O)/W type ELM.

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• Supported liquid membrane (SLM)
• preparationdip the multihole macromolecule solid
  membrane into the membrane solvent, so solvent
  can fill the holes of the membrane and form SLM.
• characteristicsimple construction, easy
  scale-up, but the solvent of membrane phase may
  be lost in use.

• Flow liquid membrane (FLM)
• also a kind of SLM, but the membrane phase is not
  easy to be lost.

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4.4.2 Extraction mechanism

• Classification

115

• Simple transfer

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• Chemical reaction of back-extraction phase
  promoting transfer

117

• Carrier transport
• Reverse direction transfer

• Parallel direction transfer

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4.4.3 Influencing factors (ELM extraction)

• The composition of membrane phase
• Membrane solvent
• Viscosity influence ELM on stability, thickness,
  and mass transfer coefficient
• There is a higher ability of dissolving the
  carrier in the case of that is necessary.
• Surfactant
• HLBVhave an effect on the stability of ELM.
  Generally the surfactants of HLB36 are used for
  preparing (W/O)/W type ELM
• Commonly nonionic surfactants, e.g. Span80, are
  applied to preparing ELM because of their
  emulsifying capacity under lower concentration
• Concentration must be suitable.
• carrier
• Only can be dissolved in membrane phase.

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• Operating condition
• pH
• Influence the extraction of weak electrolytes,
  see 4.2.1
• Mixing speed
• Influence the dispersal of emulsion and the
  stability of ELM.
• Back-extraction phase
• For the extraction process of ?and? type
  promoting transfer composition and concentration
  of back-extraction phase influence extraction
  speed and selectivity.
• Operating temperature
• Ordinarily put in practice at room temperature.
  Although extraction speeded up ELM is unstable at
  higher temperature.
• Operating time
• Extraction can be completed during short time.
  ELM is easy to be damaged if extraction time
  prolong.

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4.4.4 The processing of ELM extraction
emulsion split
ELM preparation
extraction
separation
121
4.5 Reverse micelle extraction
122

• Surfactant molecules form reverse micelles in
  organic solvents. Water can be solubilized into
  the micelles to generate water pools.
• Bioactive molecules, e.g. proteins, can be
  dissolved into those water pools, that solves the
  question of it is difficult that dissolving
  biomacromolecules in organic solvents, or organic
  solvents make the molecules denature.

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4.5.1 Reverse micelle and its basic characters

• Normal micelle
• Surfactant added into water will generate
  molecular selfassembly, and form normal micelles
  when its concentration reaches or exceeds a
  given value.
• The given value is called critical micelle
  concentration (CMC).
• Reverse micelle
• Surfactant added into organic solvent form
  reverse micelles when its concentration reaches
  or exceeds CMC.

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• Shapemajority is global or approximately
• Sizediameter is 520nm usually.
• Commonly used surfactant is AOT. The diameter of
  reverse micelles formed by AOT in isooctane can
  be calculated as

Where W0 is mole ratio between water and
surfactant, i.e. water content.
125
4.5.2 Prototype of protein solubilized
in the reverse micelle

• Four modes are presented
• For hydrophilic protein the prototype of water
  shell is generally accepted.

126
4.5.3 the factors of influencing
the extraction efficiency

• Electrostatic interaction
• Usually ionic surfactant is applied to reverse
  micelle extraction, so the inner surface of
  reverse micelle bears negative charge (AOT) or
  positive charge (TOMAC). When pH of aqueous phase
  depart the pI of protein there is a electrostatic
  interaction between protein and surfactant, that
  will influence the extraction efficiency of
  protein.
• In theory, when protein bearing the charge is
  opposite with surfactant, it is easy for protein
  to be solubilized in the reverse micelle,
  otherwise cant be dissolved.

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• Steric interaction
• If water content of reverse micelle is decreased,
  the extraction efficiency of protein is lower
• The more heavy protein molecular weight is, the
  more low the extraction efficiency of protein is.
• Hydrophobic interaction
• The hydrophobicity of protein have an effect on
  the mode of being solubilized in the reverse
  micelle, that influence its extraction
  efficiency.

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4.5.4 Operation in practice

• Multistep batch mix-clarification extraction

129

• Continue circular extraction-back extraction

130
4.6 Supercritical fluid extraction
131

• Definitionsupercritical fluid (SCF), the
  material exists as fluid above their critical
  temperature and pressure,respectively, as
  extractant is used for extracting target product
  from solid or liquid feedstock.
• ApplicationSCF have a particularly solvent
  property on fatty acid, plant alkaloid, ether,
  ketone, glycerolipid, etc. so can be used for
  their extraction.

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4.6.1 The feature of SCF

• Supercritical CO2 is most commonly used for
  extractions because of its relatively low
  critical temperature and other advantages, e.g.
  nontoxicity, higher chemical stability, and
  cheapness.

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• p-V(?)-T phase chart of CO2a tiny alter of
  temperature or pressure can generate a much more
  great change of density of CO2 near the critical
  point.
• Many of the properties of SCF are intermediate
  between those of gas and liquid, e.g. their
  diffusivity is higher than that of liquid while
  viscosity is lower.

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4.6.2 SCF extraction operation

• Isothermal operation
• Product is extracted and recovered by altering
  operating pressure
• Iso-pressure operation
• Product is extracted and recovered by altering
  operating temperature.
• Adsorption operation
• The sorbent, that can selectively adsorb target
  product, is used for recovering the product.

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Chapter 5 Membrane separation
136

• Separation principlea semi-permeable membrane
  acts as a selective barrier retaining the
  molecules/particles bigger than the pore size
  while allowing the smaller molecules to permeate
  through the pores.
• Application and advance the use of membrane
  technology for separation of biomolecules and
  particles and concentration of process fluids has
  expanded dramatically in recent years. The
  membrane function has been made more versatile by
  integrating it with other separation principles.

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5.1 Various membrane separation
processes
138
Classification
139
5.1.1 Reverse osmosis

• Reverse osmosis, or hyperfiltration, separates
  ionic solutes typically less than 1nm.
• Primarily applied to the production of pure
  water, and seawater desalination.

140
5.1.2 Microfiltration and
ultrafiltration

• Microfiltration
• Be used for separation of particles, typically
  0.0110 ?m in diameter.
• Ultrafiltration
• Separates polymeric solutes in the 0.0010.05 ?m
  range.
• Microfiltration and ultrafiltration are widely
  used in the primary recovery stages of downstream
  processing.

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5.1.3 electrodialysis

• Principleion exchange membrane is applied to
  solutes separation according to the difference of
  carrying charges and size between the molecules
  of different solutes.
• Applicationcan be used for purification of
  charged small biomolecules.

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5.1.4 Pervaporation

• Principle separation of solutes is determined by
  differences in their vapour pressure and by the
  permeability of the membrane.
• Application recovery and concentration of
  volatile products.

144
5.2 Membrane materials and membrane
peculiarities
145
5.2.1 Membrane materials

• Ideal materials should meet the expectations as
  follows
• Effective membrane thickness is thin, and pore
  density is high
• Inert materials
• Adaptable for a wide scope of pH and
  temperature
• Facile to be cleaned
• Can meet various separation destination.

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• Classification of materials
• Polymeric material
• For example cellulose acetate and polysulphone.
• Character cheap for manufacturing membrane but
  difficult to clean and may require chemical
  rather than steam sterilization.
• Inorganic material
• For example ceramics and steel.
• Character expensive for producing membrane but
  can be cleaned and sterilized in place.

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5.2.2 Constructional speciality
of membrane

• Pore structure
• Pore structures, which have an impact upon
  separation speed and capacity of resisting
  fouling, are different because of differences
  among membrane materials and manufacturing
  approaches.
• The differences between symmetric membrane and
  asymmetric membrane
• Symmetric membrane the structure of pore is
  symmetric along the direction of thickness on the
  section of membrane. The majority of
  microfiltration membrane are this type of
  structure
• Asymmetric membrane constructed with surface
  active layer (0.20.5?m) and inert layer
  (50100?m) . The majority of ultrafiltration and
  reverse osmosis membrane are asymmetric nowadays.

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• Pore property
• Parameters pore diameter, distributing of pore
  diameter, and pore density on the surface of
  membrane.
• Determination directly observe by electron
  microscope for microfiltration or ultrafiltration
  membrane.

• Generally speaking, there is a wider distribution
  range of pore diameter for almost all of the
  membrane.

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5.2.3 Important parameter of
membrane selectivity

• Molecular weight cut off (MWCO)
• Retention curve a curve, that indicate the
  correlation between retention rate (R) of
  membrane and molecular weight of solute, is
  obtained by determining the R of different
  molecular weights global proteins or
  water-solubility polymers.

• MWCO molecular weight of the solute, whose
  retention rate is 0.90 on the retention curve,
  is defined as MWCO.

150
5.3 Membrane module
151

• Commercial membrane modules

152
5.3.1 Hollow-fibre and tube
membrane module

• Hollow-fibre membrane module
• Membrane area is maximum, and the cost is cheap
  so that be commonly used. It is also easy to be
  cleaned by back-flushing.
• Tube membrane module
• The structure is simple, and it is easy to be
  cleaned but the cost is expensive.

153
5.3.2 Plate and spiry winding
membrane module

• Characteristic
• Filtration areas of the two types are great, but
  not be used widely.
• Plate membrane module is primarily used for
  microfiltration and ultrafiltraiton, and the
  other is mainly applied to reverse osmosis.

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5.4 The factors of influencing
separation velocity
155
5.4.1 Operating mode

• Traditional filtration
• Almost all are dead-end filtration, i.e. the feed
  flows on to the membrane.
• Disadvantagethe cake grows in thickness with
  time, that make the flow through the filter
  reduce.

• microfiltration and ultrafiltration
• Cross-flow filtration is available, here, a flow
  of the feed stream is maintained parallel to the
  separation surface.
• Advantagethe cake thickness is limited to a thin
  layer as compared to the dead end mode.

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5.4.2 Operating pressure

• When pressure is lower, there is a linear
  correlation between Jv and ?p

• accompanying the increase of ?p, concentration
  polarization happens on the surface of membrane,
  the correlation between Jv and ?p can be
  expressed as

• after ?p increase to the appearance of gel
  polarization, Jv is near a constant (Jlim)

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5.4.3 Velocity of flow

• Velocity of flow increasing can improve mass
  transfer coefficient k, so make Jv increase.
• In addition, velocity of flow increasing have an
  effect on weakening concentration polarization or
  gel polarization.

158
5.5 Operation in practice
159
5.5.1 Concentration

• There are three operating modes for membrane
  separation used on concentration of cell or
  protein, they are opened circuit cycling,
  closed-circuit cycling, and continuous operation.

160
5.5.2 Diafiltration

• The operating mode is applied to removal of small
  molecule solutes from high molecular solution.

161
5.6 Membrane fouling and
cleaning
162
5.6.1 Membrane fouling

• Membrane fouling is most difficult to be solved
  on application of membrane technology.
• The main reason of leading membrane fouling
• Gel layer
• The adsorption of solute on the membrane surface
• Clog of the membrane pore
• The adsorption of solute in the membrane pore.
• The measure of preventing or minimizing membrane
  fouling
• Pretreatment of membrane
• Pretreatment of feedstock

163
5.6.2 Cleaning

• Cleaning agent water, salt solution, diluted
  acid and alkali, surfactant, solution of enzyme,
  etc.
• Selection of cleaning agent
• If can select water as cleaning agent at first
• It must have an excellent detergent power
• It cant harm membrane.

164
5.7 Application
165
Membrane bioreactor

• Definition a bioreactor of coupling membrane
  separation process and bioreaction process.
• Application high-density culture of animal or
  plant cells, microbial fermentation, and enzyme
  catalysis reaction.