1

Chapter VII

Heat Transfer in Fermentation

2

Introduction

Several important chemical engineering concepts in Bioprocess Engineering are transport phenomena (fluid flow, mixing, heat and mass transfer), unit operations, reaction engineering, and bioreactor engineering.

Fluid flow, mixing, and reactor engineering are skipped in this class. They are available more detail in several chemical engineering books.

We start with the heat transfer in bioreactors

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Two types of common heat transfer application in bioreactor operation

• In situ batch sterilization of liquid medium. In this process, the fermenter vessel containing medium is heated using steam and held at the sterilization temperature for a period of time; cooling water is then used to bring the temperature back to normal operating conditions

• Temperature control during reactor operation. Metabolic activity of cells generates heat. Some microorganisms need extreme temperature conditions (e.g. psycrophilic, thermophilic microorganisms)

Heat transfer configurations for bioreactors: jacketed vessel, external coil, internal helical coil, internal baffle-type coil, and external heat exchanger.

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Pro’s and cons of the heat exchanger configurations

• External jacket and coil give low heat transfer area. Thus, they are rarely used for industrial scale.

• Internal coils are frequently used in production vessel; the coils can be operated with liquid velocity and give relatively large heat transfer area. But the coil interfere with the mixing in the vessel and make cleaning of the reactor difficult. Another problem is film growth of cells on the heat transfer surface.

• External heat exchanger unit is independent of the reactor, easy to scale up, and provide best heat transfer capability. However, conditions of sterility must be met, the cells must be able to withstand the shear forces imposed during pumping, and in aerobic fermentation, the residence time in the heat exchanger must be small enough to ensure the medium does not become depleted of oxygen.

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Heat exchangers in fermentation processes

• Double-pipe heat exchanger

• Shell and tube heat exchanger

• Plate heat exchanger

• Spiral heat exchanger

In bioprocess, the temperature difference is relatively small. Thus, plate heat exchanger is almost never being used

The concepts and calculation for heat exchangers and their configurations are available in the text book ( Pauline Doran, Bioprocess Eng Principle, chapter 8)

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Chapter VIII

Mass Transfer in Fermentation

7

Introduction

The Fick’s law of diffusion

Role of diffusion in Bioprocess

• Scale of mixing

Mixing on a molecular scale relies on diffusion as the final step in mixing process because of the smallest eddy size

• Solid-phase reaction

The only mechanism for intra particle mass transfer is molecular diffusion

• Mass transfer across a phase boundary

Oxygen transfer to gas bubble to fermentation broth, penicillin recovery from aqueous to organic liquid, glucose transfer liquid medium into mould pellets are typical example.

dy

dCDJ A

ABA

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Film theory

The two film theory is a useful model for mass transfer between phase. Mass transfer of solute from one phase to another involves transport from bulk of one phase to the interface, and then from the interface to the bulk of the second phase. This theory is based on idea that a fluid film or mass transfer boundary layer forms whenever there is contact between two phases. According to film theory, mass transfer through the film is solely by molecular diffusion and is the major resistance.

CA1i CA1 Bulk fluid 1

Bulk fluid 2 CA2i

CA2 Film 2 Film 1

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Convective mass transfer

AGiAGGAG

ALALiLAL

CCakN

CCakN

It refers to mass transfer occurring in the presence of bulk fluid motion

k: mass transfer coefficient [m/s]

a: area available for mass transfer [m2/m3]

CAo: concentration of A at bulk fluid

CAi: concentration of A at interface

For gas-liquid system, A from gas to liquid:

AiAoA CCkaN

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Overall mass transfer coefficient

Refers to the book Geankoplis (2003), Transport Processes and Separation Process Principles, 4th ed, chapter 10.4.

Oxygen transport to fermentation broth can be modeled as diffusion of A through stagnant or non-diffusing B.

If A is poorly soluble in the liquid, e.g. oxygen in aqueous solution, the liquid-phase mass transfer resistance dominates and kGa is much larger than kLa. Hence, KLa ≈ kLa.

ALALLA

LGG

CCaKN

ak

m

akaK

*

'11

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Oxygen transfer from gas bubble to cell

Eight steps involved:i. Transfer from the interior of the bubble to the gas-liquid interface

ii. Movement across the gas-liquid interface

iii. Diffusion through the relatively stagnant liquid film surrounding the bubble

iv. Transport through the bulk liquid

v. Diffusion through the relatively stagnant liquid film surrounding the cells

vi. Movement across the liquid-cell interface

vii. If the cells are in floc, clump or solid particle, diffusion through the solid of the individual cell

viii. Transport through the cytoplasm to the site of reaction.

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Analyzes for most bioreactors in each step involved

i. Transfer through the bulk phase in the bubble is relatively fastii. The gas-liquid interface itself contributes negligible resistanceiii. The liquid film around the bubble is a major resistance to oxygen

transferiv. In a well mixed fermenter, concentration gradients in the bulk liquid

are minimized and mass transfer resistance in this region is small, except for viscous liquid.

v. The size of single cell <<< gas bubble, thus the liquid film around cell is thinner than that around the bubble. The mass transfer resistance is negligible, except the cells form large clumps.

vi. Resistance at the cell-liquid interface is generally neglectedvii. The mass transfer resistance is small, except the cells form large

clumps or flocs.viii. Intracellular oxygen transfer resistance is negligible because of the

small distance involved

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Chapter IX

Unit Operations in Fermentation

(introduction to downstream processing)

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Downstream processing, what and why

Downstream processing is any treatment of culture broth after fermentation to concentrate and purify products. It follows a general sequence of steps:

1.Cell removal (filtration, centrifugation)

2.Primary isolation to remove components with properties significantly different from those of the products (adsorption, liquid extraction, precipitation). Large volume, relatively non selective

3.Purification. Highly selective (chromatography, ultra filtration, fractional precipitation)

4.Final isolation (crystallization, followed by centrifugation or filtration and drying). Typical for high-quality products such as pharmaceuticals.

Downstream processing mostly contributes 40-90 % of total cost.

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Filtration

Type of filtration unit:

• Plate and frame filter. For small fermentation batches

• Rotary-drum vacuum filter. Continuous filtration that is widely used in the fermentation industry. A horizontal drum 0.5-3 m in diameter is covered with filter cloth and rotated slowly at 0.1-2 rpm.

The filtration theory and equation are not explained here since they are available in the course “Unit Operations of Chemical Engineering I”.

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Centrifugation

Centrifugation is used to separate materials of different density when a force greater than gravity is desired

The type of industrial centrifugation unit:

• Tubular bowl centrifuge (Narrow tubular bowl centrifuge or ultracentrifuge, decanter centrifuge, etc). Simple and widely applied in food and pharmaceutical industry. Operates at 13000-16000 G, 105-106 G for ultracentrifuge

• Disc-stack bowl centrifuge. This type is common in bioprocess. The developed forces is 5000-15000 G with minimal density difference between solid and liquid is 0.01-0.03 kg/m3. The minimum particle diameter is 5 µm

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Centrifugation (dry solid decanter centrifuge)

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The centrifugation theory

gDu pfp

g2

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The terminal velocity during gravity settling of a small spherical particle in dilute suspension is given by Stoke’s law:

Where ug is sedimentation velocity under gravity, ρp is particle density, ρf is liquid density, µ is liquid viscosity, Dp is diameter of the particle, and g is gravitational acceleration.

In the centrifuge:

uc is particle velocity in the centrifuge, ω is angular velocity in rad/s, and r is radius of the centrifuge drum.

rDu pfp

c22

18

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The centrifugation theory

g

rZ

2

gu

Q

2

The ratio of velocity in the centrifuge to velocity under gravity is called the centrifuge effect or G-number.

Industrial Z factors: 300-16 000, small laboratory centrifuge may up to 500 000. The parameter for centrifuge performance is called Sigma factor

Q is volumetric feed rate. The Sigma factor explain cross sectional area of a gravity settler with the same sedimentation characteristics as the centrifuge. If two centrifuge perform with equal effectiveness

2

2

1

1

QQ

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The centrifugation theory 3

13

2

2

tan3

12rr

g

N

Disc-stack bowl centrifuge

N is number of disc, θ is half-cone angle of the disc.

The r1 and r2 are inner and outer radius of the disc, respectively.

Tubular-bowl centrifuge 21

22

2

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rrg

b

b is length of the bowl, r1 and r2 are inner and outer radius of the wall of the bowl.

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Cell disruption

Mechanical cell disruption methods

•French press (pressure cell) and high-pressure homogenizers. In these devices, the cell suspension is drawn through a check valve into a pump cylinder. At this point, it is forced under pressure (up to 1500 bar) through a very narrow annulus or discharge valve, over which the pressure drops to atmospheric. Cell disruption is primary achieved by high liquid shear in the orifice and the sudden pressure drop upon discharge causes explosion of the cells.

•Ultrasonic disruption. It is performed by ultrasonic vibrators that produce a high-frequency sound with a wave density of approximately 20 kHz/s. A transducer convert the waves into mechanical oscillations via a titanium probe immersed in the concentrated cell suspension. For small scale

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Cell disruption

                        

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The equation for Manton-Gaulin homogenizer

kNpRR

R

m

m

ln

Rm: maximum amount protein available for release

R: amount of protein release after N passes through the homogenizer

k: temperature-dependent rate constant

p: operating pressure drop

: resistance parameter of the cells, for S. cerevisiae is 2.9

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Cell disruption

Non mechanical cell disruption methods

Autolysis, use microbe own enzyme for cell disruption

Osmotic shock. Equilibrating the cells in 20% w/v buffered sucrose, then rapidly harvesting and resuspending in water at 4oC.

Addition of chemicals (EDTA, Triton X-100), enzymes (hydrolyses, -glucanases), antibiotics (penicillin, cycloserine)

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Chromatography

Chromatographic techniques usually employed for high value products. These methods, normally involving columns of chromatographic media (stationary phase), are used for desalting, concentration and purification of protein preparations. Several important aspects are molecular weight, isoelectric point, hydrophobicity and biological affinity. The methods are:

1.Adsorption chromatography

2.Affinity chromatography

3.Gel filtration chromatography

4.High performance liquid chromatography

5.Hydrophobic chromatography

6.Metal chelate chromatography

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Finishing steps (final isolation)

Crystallization

Product crystallization may be achieved by evaporation, low-temperature treatment or the addition of a chemical reactive with the solute. The product’s solubility can be reduced by adding solvents, salts, polymers, and polyelectrolytes, or by altering pH.

Drying

Drying involves the transfer of heat to the wet material and removal of the moisture as water vapor. Usually, this must be performed in such a way as to retain the biological activity of the product. The equipment could be rotary drum drier, vacuum tray drier, or freeze-drier.

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Chapter X

Bioreactor

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Bioreactor configurations

Stirred tank bioreactor

Similar to CSTR; this requires a relatively high input of energy per unit volume. Baffles are used to reduce vortexing. A wide variety of impeller sizes and shapes is available to produce different flow patterns inside the vessel; in tall fermenters, installation of multiple impellers improves mixing.

Typically, only 70-80 % of the volume of stirred reactors is filled with liquid; this allows adequate headspace for disengagement of droplets from exhaust gas and to accommodate any foam which may develop. Foam breaker may be necessary if foaming is a problem. It is preferred than chemical antifoam because the chemicals reduce the rate of oxygen transfer.

The aspect ratio (H/D) of stirred vessels vary over a wide range. When aeration is required, the aspect ratio is usually increased. This provides for longer contact times between the rising bubbles and liquid and produces a greater hydrostatic pressure at the bottom of the vessel.

Care is required with particular catalysts or cells which may be damaged or destroyed by the impeller at high speeds.

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Bioreactor configurations

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Bioreactor configurations

Bubble column

In bubble-column reactors, aeration and mixing are achieved by gas sparging; this requires less energy than mechanical stirring. Bubble columns are applied industrially for production of bakers’ yeast, beer and vinegar, and for treatment of wastewater.

A height-to-diameter ration of 3:1 is common in bakers’ yeast production; for other applications, towers with H/D of 6:1 have been used. The advantages are low capital cost, lack of moving parts, and satisfactory heat and mass transfer performance. Foaming can be problem.

Homogeneous flow: all bubbles rise with the same upward velocity and there is no back-mixing of the gas phase.

Heterogeneous flow: At higher gas velocity. Bubbles and liquid tend to rise up in the center of the column while a corresponding down flow of liquid occurs near the walls.

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Bioreactor configurationsAirlift reactor

Airlift reactors are often chosen for culture of plant and animal cells and immobilized catalyst because shear level are low. Gas is sparged into only part of the vessel cross section called the riser. Gas hold-up and decreased liquid fluid density cause liquid in the riser to move upwards. Gas disengages at the top of the vessel leaving heavier bubble-free liquid to recirculate through the downcomer. Airlift reactors configurations are internal-loop vessels and external-loop vessels. In the internal-loop vessels, the riser and downcomer are separated by an internal baffle or draft tube. Air may be sparged into either the draft tube or the annulus. In the external-loop vessels, separated vertical tubes are connected by short horizontal section at the top and bottom. Because the riser and downcomer are further apart in external-loop vessels, gas disengagement is more effective than in internal-loop devices. Fewer bubbles are carried into the downcomer, the density difference between fluids in the riser and downcomer is greater, and circulation of liquid in the vessel is faster. Accordingly, mixing is usually better in external-loop than internal-loop reactors.

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Bioreactor configurations

33

Stirred and air-driven reactors: comparison of operating characteristic

For low-viscosity fluids, adequate mixing and mass transfer can be achieved in stirred tanks, bubble columns and airlift vessels. When a large fermenter (50-500 m3) is required for low-viscosity culture, a bubble column is an attractive choice because it is simple and cheap to install and operate. Mechanical-agitated reactors are impractical at volumes greater than about 500 m3 as the power required to achieve adequate mixing becomes extremely high.

Stirred reactor is chosen for high-viscosity culture. Nevertheless, mass transfer rates decline sharply in stirred vessels at viscosities > 50-100 cP.

Mechanical-agitation generates much more heat than sparging of compressed gas. When the heat of reaction is high, such as in production of single cells protein from methanol, removal of frictional stirrer heat can be problem so that air-driven reactors may be preferred.

Stirred-tank and air-driven vessels account for the vast majority of bioreactor configurations used for aerobic culture. However, other reactor configurations may be used in particular processes

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Other bioreactors

Packed bed

Used with immobilized or particulate biocatalysts, for example during the production of aspartate and fumarate, conversion of penicillin to 6-aminopenicillanic acid, and resolution of amino acid isomers. Damaged due to particle attrition is minimal in packed beds compared with stirred reactors.

Mass transfer between the liquid medium and solid catalyst is facilitated at high liquid flow rate through the bed. To achieve this, packed are often operated with liquid recycle. The catalyst is prevented from leaving the columns by screens at the liquid exit. Aeration is generally accomplished in a separated vessel because if air is sparged directly into the bed, bubble coalescence produces gas pockets and flow channeling or misdistribution. Packed beds are unsuitable for processes which produce large quantities of carbon dioxide or other gases which can become trapped in the packing.

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Other bioreactors

Fluidized bed

To overcome the disadvantages of packed bed, fluidized bed may be preferred. Because particles are in constant motion, channeling and clogging of the bed are avoided and air can be introduced directly into the column. Fluidized bed reactors are used in waste water treatment with sand or similar material supporting mixed microbial populations, and with flocculating organisms in brewing and production of vinegar.

Trickle bed

Is another variation of the packed bed. Liquid is sprayed onto top of the packing and trickles down through the bed in small rivulets. Air may be introduced at the base; because the liquid phase is not continuous throughout the column, air and other gases move with relative ease around the packing. Trickle-bed reactors are used widely for aerobic wastewater treatment.

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Other bioreactors