Bài giảng Kỹ thuật phản ứng sinh học: Chương 5 - Bùi Hồng Quân

Bài giảng Kỹ thuật phản ứng sinh học: Chương 5 Quá trình truyền khối khí lỏng, cung cấp cho người học những kiến thức như: Khái niệm cơ bản; Tính toán quá trình truyền khối khí lỏng. Mời các bạn cùng tham khảo!

Chương 5 Quá trình truyền khối khí lỏng

 5.1 Khái niê ̣m cơ bản

 5.2 Tính toán quá trình truyền khói khí lỏng

BIOREACTORS BIOREACTORS

 Importance of considering process engineering factors when culturing cells

 Biological factors include the characteristics of the cells, their maximum specific growth rate, yield coefficient, pH range and temperature range

 The productivity of a fermentation is determined by the mode of operation of the fermentation process; eg the advantages of fed-batch and continuous

 The oxygen demand of an industrial process is generally satisfied by aeration and agitation

 Productivity is limited by oxygen availability and therefore it is important to the factors that affect a fermenters efficiency in supplying O2

 O2 requirement, quantification of O2 transfer and factors influencing the transfer of O2 into solution

Bioreactors Bioreactors Introduction Introduction

Bioreactors- Introduction

Mass transfer, in particular, oxygen transfer are important factor which determined how a reactor must be designed and operated

Cost was also described as an important consideration The larger the reactor or the faster the stirrer speed, the greater the costs involved

The rate of oxygen transfer = driving force / resistance E.g resistance to mass transfer from medium to mo`s are complex and may arise from;

 Diffusion from bulk gas to gas/liquid interface

 Solution of gas in liquid interface

 Diffusion of dissolved gas to bulk of liquid

 Transport of dissolved gas to regions of cell

 Diffusion through stagnant region of liquid surrounding the

cell

 Diffusion into cell

 Consumption by organism (depends on growth/respiration

kinetics)

phases and material that are relevant in general transport processes associated with fermentation technology;

Phases present in bioreaction / bioreactor

Non aqueous phase Aqueous phase Solid phase

(Reactants / products) Dissolved reactants /

products

Reaction

Liquids (e.g oils) Sugars Organelles

Solid (e.g particles of

substrate) Minerals Enzymes

• One of the most critical factors in the operation of a fermenter is the

provision of adequate gas exchange

•The majority of fermentation processes are aerobic

• Oxygen is the most important gaseous substrate for microbial

metabolism, and carbon dioxide is the most important gaseous metabolic product

• For oxygen to be transferred from a air bubble to an individual microbe,

several independent partial resistance’s must be overcome

Stoichiometry of respiration

To consider the Stoichiometry of respiration the oxidation

of glucose may be represented as;

C6H12O6 + 6O2 = 6H2 O + 6CO2

Atomic weight of Carbon

Hydrogen Oxygen

12

1

16

Molecular weight of glucose is 180

How many grams of oxygen are required to oxidise 180g of glucose ?

Solubility of Oxygen

 Both components oxygen and glucose must be in solution before they become available to microorganisms

 Oxygen is 6000 times less soluble in water than glucose

 A saturated oxygen solution contains only10mg dm -3 of oxygen

 Impossible to add enough oxygen to a microbial culture to satisfy needs for complete respiration

 Oxygen must be added during growth at a sufficient rate to satisfy requirements

glucose and oxygen by yeast

Problems encountered in oxygen transport can be illustrated

by comparing transport of glucose vs oxygen;

1% Sugar (glucose) Broth O 2 sat @ 25 o C

Conc in bulk broth 10,000 ppm approx 7 ppm

Critical conc 100 ppm 0.8 ppm

(growth stops)

Rate of demand 2.8 mmoles/ g cells /h 7.7 mmoles/

g cells /h

MASS TRANSFER and RESPIRATION

(a) Mass balance

Stoichiometry of respiration e.g glucose;

C 6 H 12 O 6 + 6O 2  6H 2 O + 6 CO 2

Oxidation of 180 gms Glucose requires 192 gms O 2

Compare with a hydrocarbon (i.e 6 CH 2 )

The Oxygen requirements of industrial

Compare solubility of Oxygen vs Glucose ( e.g oxygen = 9.0 mg/l @ 20oC, 11.3 mg/l @ 10oC)

Thus must consider;

Requirement for oxygen important in biotechnological processes

Quantification of oxygen transfer (to avoid rate limiting step) important

 Factors influencing rate of transfer (e.g viscosity) important

FACTORS AFFECTING OXYGEN DEMAND

 Rate of cell respiration

 Type of respiration (aerobic vs anaerobic)

 Type of substrate (glucose vs methane)

 Type of environment (e.g pH, temp etc.)

 Surface area/ volume ratio

large vs small cells (bacteria v mammalian cells) hyphae, clumps, flocks, pellets etc

 Nature of surface area (type of capsule etc)

Methods of Aeration

microorganisms They vary in size and complexity from a

10 ml volume in a test tube to computer controlled

similarly vary in cost from dollars to a few million dollars

reactors

 Standing cultures

 Shake flasks

 Stirred tank reactors

 Bubble column and airlift reactors

 Fluidized bed reactors

In standing cultures, little or no power is used for aeration Aeration is dependent on the transfer of oxygen through the still surface of the culture

Standing cultures

The rate of oxygen transfer will be poor due to the small surface area for transfer Standing cultures are commonly used in small scale laboratory systems in which oxygen supply is not critical For example, biochemical tests used for the identification of bacteria are often performed in test-tubes containing between 5-10 ml of media

T-flasks used in the small scale culture of animal cells are another example of a standing culture T-flasks are normally incubated horizontally to increase the surface area for oxygen transfer.

Standing cultures

• Large Pyrex flasks are used for the small scale production

of fermented products

• Standing culture aeration is not restricted to the laboratory

• In some countries, where the availability of electricity is

unreliable, citric acid is produced using surface culture

techniques

• In these cultures, the Aspergillus niger mycelia are grown

on the surface of liquid media in large shallow trays

• The medium is neither gassed nor agitated

Aspergillus niger mycelia

Standing cultures

Aerobic solid substrate fermentations are another example

of standing cultures In these fermentations, the biomass is grown on solid biodegradable substrates

The solids may be continuously or periodically turned over

to improve aeration and to regulate the culture temperature One example of a commercial scale, solid substrate fermentation is the production of koji by

Aspergillus oryzae on soya beans which is part of the soya

sauce process

Another is mushroom cultivation Considerable research is currently being invested into the feasibility of producing biochemicals by solid substrate fermentations

Shake flasks

Shaking continually breaks the liquid surface and thus provides a greater surface area for oxygen transfer

Increased rates of oxygen transfer are also achieved by entrainment of oxygen bubbles at the surface of the liquid

Shake flasks

Although higher oxygen transfer rates can be achieved with shake flasks than with standing cultures, oxygen transfer limitations will still be unavoidable particularly when trying to achieve high cell densities

The rate of oxygen transfer in shake flasks is dependent on the

 shaking speed

 the liquid volume

 shake flask design

Shake flasks O2 Transfer

k L a decreases with liquid volume k L a is higher when baffles are present

Shake flasks O2 Transfer

The kLa will increase with the shaking speed

At high shaking speeds, bubbles become entrained into the medium to further increases the oxygen transfer rate

The presence of baffles in the flasks will further increase the oxygen transfer efficiency, particularly for orbital shakers

The following photographs show how baffles increase the level of gas entrainment in a shake flask being shaken in an orbital shaker at 150 rpm

Unbaffled flask Baffled flask

Shake flasks O2 Transfer

due to the higher level of gas entrainment

with horizontal reciprocating shakers

volume For example, for a standard 250ml flask, the liquid volume should not exceed 70 ml while for a 1 litre flask, the liquid volume should be less than 200 ml

Mechanically stirred bioreactors

Mechanically stirred bioreactors

For aeration of liquid volumes greater than 200 ml, various options are available

Non-sparged mechanically agitated bioreactors can supply sufficient aeration for microbial fermentations with liquid volumes up to 3 litres

However, stirring speeds of up to 600 rpm may be required before the culture is not oxygen limited

In non-sparged reactors, oxygen is transferred from the space above the fermenter liquid Agitation continually breaks the liquid surface and increases the surface area for oxygen transfer.

head-stirred tank bioreactors

required for effective oxygen transfer

sparging, leads to a dramatic increase in the oxygen transfer area

agitation speeds for aeration efficiencies comparable

to those achieved in non-sparged fermenters

greater than 500,000 litres

Bubble driven bioreactors

aeration and agitation Two classes of bubble driven

of shear sensitive organisms such as moulds and plant cells

An airlift fermenter differs from bubble column bioreactors

by the presence of a draft tube which provides better mass and heat transfer efficiencies

to construct than bubble column reactors There are several designs for air-lift fermenters although the most commonly

Bubble driven bioreactors

bioreactors by the presence of a draft tube which provides

 better mass and heat transfer efficiencies

 more uniform shear conditions

height to base ratios of between 8:1 and 20:1

hold-ups, long bubble residence times and a region of high hydrostatic pressure near the sparger at the base of the fermenter

enhanced oxygen transfer rates

An airlift fermenter differs from bubble column bioreactors by the presence of a draft tube

The presence of the draft tube enhances axial mixing throughout the whole reactor

This presumably occurs due to circulatory effect that the draft tube induces in the reactor The circulation occurs in one direction and hence the bubbles also travel in one direction

Airlift bioreactors

Small bubbles lead to an increased surface area for oxygen transfer

Equalize shear forces throughout the reactor Major reason why the productivity of cells grown in airlift bioreactors have higher productivities than those grown in stirred tank reactors

Airlift bioreactors

The major disadvantages of air-lift fermenters are

 high energy requirements

Airlift bioreactor

Air-riser and down-comer

 An air-lift reactor is divided into three regions:

- the air-riser

- down-comer

- disengagement zone

Airlift bioreactor

The region into which bubbles are sparged is called the

air-riser The air-riser may be on the inside or the outside

of the draft-tube The latter design is preferred for large scale fermenters as it provides better heat transfer efficiencies

in a vertical direction To counteract these upward forces,

liquid will flow in a downward direction in the

down-comer This leads to liquid circulation and thus improved

mixing efficiencies as compared to bubble columns

move in a uniform direction at a relatively uniform velocity This bubble flow pattern reduces bubble

compared to bubble column reactors

Airlift bioreactors - Disengagement zone

Airlift bioreactors - Disengagement zone

 The roles of the disengagement zone are to

 add volume to the reactor,

 reduce foaming and

 minimise recirculation of bubbles through the down comer

Airlift bioreactors - Disengagement zone

the bubble velocity and thus disengages the bubbles from the liquid flow

entering the downcomer

zone also leads to a reduction in the loss of medium due aerosol formation

in foams, causing the bubbles to burst The axial flow circulation caused by the draft tube also helps to reduce foaming

Packed bed and trickle flow bioreactors

The topic of packed bed bioreactors was discussed in another lecture on immobilisation

Packed bed bioreactors

depends on the flow rate and on the thickness of the biomass film on or near the surface of the solid particles

mass transfer rates and clogging Despite this they are used commercially with enzymatically catalysts and with slowly or non-growing cells

wastewaters (eg food processing wastes) Large plastic blocks are used as solid supports for the cells These blocks have a large surface area for cell immobilization and when packed in the reactor are difficult to clog

Trickle flow bioreactors

Trickle bed reactors are a class of packed bed reactors in which the medium flows (or trickles) over the solid particles In these reactors, the particles are not

The liquid medium trickles over the surface of the

solids on which the cells are immobilized

Oxygen transfer is enhanced by ensuring that the cells are covered by only a very thin layer of liquid, thus reducing the distance over which the dissolved oxygen must diffuse to reach the cells

Trickle flow bioreactors

Because stirring is not used, considerable capital costs are saved

However, oxygen transfer rates per unit volume are low compared with spared stirred tank systems

Trickle flow systems are used widely for the aerobic treatment of sewage

They are used to polish effluent from the activated sludge

or anaerobic digestion process and for the nitrification of ammonia.

Fluidized bed reactors

Fluidized bed reactors

 Fluidised bed bioreactors are one method of maintaining high biomass concentrations and at the same time good mass transfer rates in continuous cultures

 Fluidised bed bioreactors are an example of reactors in which mixing is assisted by the action of a pump In a fluidised bed reactor, cells or enzymes are immobilised in and/or on the surface of light particles

 A pump located at the base of the tank causes the immobilised catalysts to move with the fluid The pump pushes the fluid and the particles in a vertical direction The upward force of the pump is balanced by the downward movement of the particles due to gravity This results in good circulation.

Fluidised bed reactors

For aerobic microbial systems, sparging is used to improve oxygen transfer rates

A draft tube may be used to improve circulation and oxygen transfer Both aerobic and anaerobic fluidised bed bioreactors have been developed for use in waste treatment

Fluidised beds can also be used with microcarrier beads used in attached animal cell culture

Fluidised-bed microcarrier cultures can be operated both

in batch and continuous mode In the former the