Ghasem D. Najafpour. Biochemical Engineering and Biotechnology

Подождите немного. Документ загружается.

164 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY

The gas superficial velocity is:

(E.1.14)

The mass transfer coefficient is defined as turbulent:

(E.1.15)

Gas hold-up is defined as volume of gas per unit volume of reactor. For air in water, Richard’s

data are by:

(E.1.16)

(E.1.17)

H ⫽ 1.94 m for aeration

(E.1.18)

Gas hold-up ⫽ 23%.

Example 2

The Monod rate model is valid for a CSTR bioreactor with maximum specific growth rate

of 0.5 h

⫺1

and K

2g⭈l

⫺1

. What would be a suitable dilution rate at steady-state condition,

where there is no cell death if initial substrate concentration is 50 g⭈l

⫺1

and yield of bio-

mass on substrate is 100%.

⫽

⫹

⫽

⫹

⫽

194

194 65

023

180

81 68

26 60 763 237

04 05

min

⫻⫽⫹

763 237

() . .

⫽⫹

Gas hold-up,

Bubble volume

Reactor volume

H ⫽

⫽⫻ ⫽

⫺⫺

210 1097

0 667 1

⫽⫽

32 67

()

␲

min

Ch006.qxd 10/27/2006 10:44 AM Page 164

BIOREACTOR DESIGN 165

Solution

The Monod rate is

(E.2.1)

x/s

⫽ 1

⫽ 50 g.l

⫺1

Substrate balance:

Assume no death rate,

Use (6.34) and rearrange to get DX:

Take derivative to obtain a value for maximum dilution rate:

max max

()

⫽⫺

⫹

⫽⫺ ⫽

⫽

⫺

m 11

0.5 0.4 h

⫺

⫺⫺

⫺

KDKD

Sm S

()

dd()DX DⲐ⫽0

DS S

KSY

DX Y S

Sxs

()

max

in out

⫺⫽

⫹

⫽⫺

⫺

ËË

qXYSS

pxs

⫽⫽⫺0

in out

,()

steady state

,⫺⫽ ⫽ ⫽

D0, m

⫺⫽ ⫺ ⫺ ⫺

in out

()

⫽

⫹

⫽

⫹

⫽

⫺

max max

max

out

Ch006.qxd 10/27/2006 10:44 AM Page 165

166 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY

Get

Then calculate the substrate concentration at leaving stream:

Example 3

A 20 m

working volume of a bioreactor is used for producting penicillin. What would be

the sugar concentration (S

) you choose if oxygen transfer rate is not the limiting reactant?

Given data:

Impeller speed ⫽ 1.5 rps (90 rpm)

Number of blades ⫽ 8; flat, turbine types of blade

m ⫽ 1mPa⭈s

r ⫽ 1.2 ⫻ 10

kg/m

Aeration rate ⫽ 1 vvm

Ratio of gassed to ungassed power, P

/P ⫽ 0.4

Driving force for OTR⫽ 6 ⫻ 10

⫺3

kg/m

Specific O

uptake ⫽ 0.65 mmol O

/kg cell

Also, the kinetic data are given as ␯

max

⫽ 0.5 h

⫺1

Specific sugar consumption rate of cells⫽ 1.0 kg/kg cell⭈h

Solution

Given data:

tank

⫽ 2.4 m

Data:

tank

⫽ 2.4 m

⫽ D

tank

/3 ⫽ 0.8 m; for three sets of impellers

Impeller speed 150 rpm; assume broth viscosity is 1 cp and the specific gravity of the broth

is 1.2; aeration rate is 1 vvm; given ratio of gassed power to un-gassed system is 0.4; spe-

cific oxygen uptake is 0.65 mmol O

/kg cell; OTR ⫽ 6 ⫻ 10

⫺3

kg/m

tank

⫽ 3

⫽ 0.8 m, three sets of impellers are used.

m ⫽ 1 mpas

r ⫽ 1200 kg/m

/P ⫽ 0.4, v

max

⫽ 0.5 h

⫺1

Specific sugar consumption rate of cells⫽ 1.0 kg (kg cell)

⫺1

⭈h

⫺1

out

g/l⫽

⫺

⫽

⫺

⫽

max

(.)

204

05 04

max max

⫽⫺

⫹

m 1

Ch006.qxd 10/27/2006 10:44 AM Page 166

BIOREACTOR DESIGN 167

Mass transfer is calculated by the empirical correlation defined for non-Newtonian filamen-

tous fermentation:

Read power number versus Reynolds number in turbulent region is based on geometry of the

impellers. The lowest power number is less than 1,for marine propellers. For flat bladed tur-

bines in a turbulent region, the power number is equal to 6. The power graph is illustrated

in Figure 6.6.

for three sets of impelers, 15 hp

The Correction factor for non-geometrical similarity is:

⫽⫽

⫻

⫽

3442

1225

P ⫽⫽()()()three sets 1.25 5.04 19 hp

hp⫽⫽0 4 15 6.( )

P ⫽⫽

3758

745 7

PN D

⫽⫽

⫻⫻

6 1200 2 5 0 8

Ungassed power,

(.)(.

))

981

3758⫽

kg•m

Ka S

⫽⫻

⫽

⫺

210

0 667

Gassed power

volume

⫽⫽

⫽

gas superficial velocity,

min

Ch006.qxd 10/27/2006 10:44 AM Page 167

168 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY

For the agitated and aerated vessel, the ratio of power requirements for aerated versus non-

aerated systems is expressed by a dimensionless number known as the aeration rate; the value

is obtained from Figure 6.7.

Maximum cell concentration, OTR ⫽ x qO

6.11 NOMENCLATURE

Rate of product formation, g⭈l

⫺1

⭈h

⫺1

⫺r

Rate of substrate consumption, g⭈l

⫺1

⭈h

⫺1

Fr Froude number, dimensionless

g gravity, m/s

N rotational speed, Hz

kg O

kg cell s

0 65 10 32 10 2 08 10

403

33 3

⫽⫻ ⫻ ⫽⫻

⫽⫽

⫻

⫺⫺ ⫺

(. )( ) .

110

208 10

19 375

⫺

⫻

⫽

⫽⫹ ⫽⫹

⫽

38 75⫽

min

⫽

⫽⫽

⫽

0 333

25 08

026

20 0 333

(.)(.)

4419 7

442 74 10

()

(.)

⫽

⫽⫽⫽⫻

⫽

⫺

.6 hp

min

␲

⫻⫻⫽⫻

⫽⫺

⫺⫺

442 67 10

0 667 2

(. ) .

()

OTR K a C C

OTR

⫽⫽⫽⫻ ⫻ ⫽⫻

⫺⫺ ⫺

(. )( ) .6 7 10 6 10 4 03 10

23 4

Ch006.qxd 10/27/2006 10:44 AM Page 168

BIOREACTOR DESIGN 169

Impeller diameter, m

Power number, dimensionless

Gas superficial velocity, cm⭈min

⫺1

Dimensionless aeration rate

REFERENCES

1. Demain, A.L. and Solomon, A.N., Sci. Am. 245, 67 (1981).

2. Aiba, S. Humphrey, A.E. and Millis, N.F., “Biochemical Engineering”, 2nd edn. Academic Press, New York,

1973.

3. Phaff, H.J., Sci. Am. 245, 77 (1981).

4. McCabe, W., Smith, J. and Harriott, P., “Unit Operations of Chemical Engineering”, 6th edn. McGraw-Hill,

New York, 2000.

5. Wang, D.I.C., Cooney, C.L., Deman, A.L., Dunnill, P., Humphrey, A.E. and Lilly, M.D., “Fermentation and

Enzyme Technology”. John Wiley & Sons, New York, 1979.

6. Baily, J.E. and Ollis, D.F., “Biochemical Engineering Fundamentals,” 2nd edn. McGraw-Hill, New York, 1986.

7. Doran, P.M., “Bioprocess Engineering Principles”. Academic Press, New York, 1995.

8. Scragg, A.H., “Bioreactor in Biotechnology, A Practical Approach”. Ellis Horwood Series in Biochemistry

and Biotechnology, New York, 1991.

9. Blakebrough, N., McManamey, W.J. and Tart, K.R., Trans. Inst. Chem. Eng 56 127 (1978).

10. Shuler, M.L. and Kargi, F., “Bioprocess Engineering, Basic Concepts”. Prentice Hall, New Jersey, 1992.

11. Ghose, T.K., “Bioprocess Computation in Biotechnology,” vol. 1. Ellis Horwood Series in Biochemistry and

Biotechnology, New York, 1990.

Ch006.qxd 10/27/2006 10:44 AM Page 169

CHAPTER 7

Downstream Processing

7.1 INTRODUCTION

Bioprocessing treats raw materials and generates useful products. Most bioprocesses involve

one or more of the following processes: centrifugation, chromatography, cooling, crystallisa-

tion, dialysis, distillation, drying, evaporation, filtration, heating, humidification, membrane

separation, milling, mixing, precipitation, centrifugation, solid handling and solvent extraction.

A particular sequence of unit operations and bioprocesses is used for the manufacture of an

extra pure pharmaceutical product.

Fermentation broths are complex, aqueous mixtures of cells, comprising soluble extracel-

lular, intracellular products and any unconverted substrate or unconvertible components.

Recovery and extraction of product is important in bioprocess engineering. In particular, sep-

aration is a useful technique; it depends on product, its solubility, size of the process, and prod-

uct value. Purification of high-value pharmaceutical products using chromatography such as

hormones, antibody and enzymes is expensive and difficult to scale up.

The necessary steps

to follow a specific process depend on the nature of the product and the characteristics of the

fermentation broth. There are a few steps for product recovery; the following processes are dis-

cussed, which are considered as an alternative for product recovery from fermentation broth.

Initially fermentation broth has to be characterised on the viscosity of the fluid. If the pres-

ence of the biomass or cells causes trouble, they have to be removed. The product is stored

inside the cells, the cells must be ruptured and the product must be freed. Intracellular protein

can easily be precipitated, settled or filtered. In fact the product in diluted broth may not be

economical enough for efficient recovery. Enrichment of the product from the bioreactor efflu-

ents for increasing product concentration may reduce the cost of product recovery. There are

several economical methods for pure product recovery, such as crystallisation of the product

from the concentrated broth or liquid phase. Even small amounts of cellular proteins can be

lyophilised or dried from crude solution of biological products such as hormone or enzymes.

2,3

7.2 DOWNSTREAM PROCESSING

In downstream processing of a fermentation unit for enzyme production with a feed stream

of sugar at a concentration of 35 gl

1

, the expected product to be recovered is -amylase.

170

Ch007.qxd 10/27/2006 10:44 AM Page 170

DOWNSTREAM PROCESSING 171

The concentration of enzyme is very low, about several hundred milligrams per litre in the fer-

mentation broth. Solvent extraction is a suitable process to recover a small amount of enzyme.

The chance of some enzyme being intracellular is high, therefore cells are ruptured to liber-

ate enzyme, which can then interact with organic solvents. Figure 7.1 shows a simple diagram

for a jacketed fermentation vessel for operation at constant temperature.

Bioprocessing includes the bioreactor and a subsequent section for product recovery. The

particular separation techniques useful for any given bioprocess depend not only on the size,

charge and solubility of the product, but also on the size of the process and product value.

For example, various forms of gel filtration, gel chromatography and ion exchanger are used

to purify highly valuable pharmaceutical biological compounds such as hormones, antibi-

otics and enzymes. This chapter emphasises product recovery from fermentation products.

Our discussion on separation and purification will cover several bioprocesses, such as solvent

extraction and recovery of whole or part of the microbial products. The ultimate challenge is

to select the best combination of substrate, enzyme or organism, bioreactor and separation

of the specific product. Each separation needed depends on initial broth characteristics such

as viscosity, product concentration, impurities and undesired particulates, and final product

concentration needed for crystallisation, concentrated liquid product or dried powder.

It is often desirable to recover product and to choose a suitable strain of microorganism

which produces extracellular rather than intracellular product. If the product stays inside the

cells, the cells must be ruptured, so freeing intracellular enzyme, after which extraction or

purification is performed to recover the valuable product. The fermentation broth has to be

processed, and pass through several stages for separation and purification. The product requires

a sequence of operations for high purification. The usual steps to follow are as follows.

(1) Removal of insoluble particulates using various separation techniques. Common opera-

tions are filtration, centrifugation and/or settling/sedimentation/decanting.

(2) Primary isolation is done to increase product concentration. Solvent extraction, absorption,

precipitation and ultrafiltration are the best known. Ultrafiltration can discriminate at the

molecular level. During primary isolation, desired product concentration increases con-

siderably and substances of widely differing polarities are separated from the product.

(3) Product purification. For production of highly pure product the impurities have to be

removed for further product concentration such as chromatography and adsorption.

Feed stream contains 35g/l sugar

α-amylase

Cells as biomass in the broth

out

FIG. 7.1. Schematic of jacketed fermentation vessel.

Ch007.qxd 10/27/2006 10:44 AM Page 171

172 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY

These operations often select for impurity removal as well as further product concentra-

tion. Approaches include fractional precipitation. Other alternatives such as chromato-

graphy and adsorption are also considered as methods of process purification.

(4) Final product isolation and drying of the crystallised product are done by drum drying,

spray drying or freeze drying. The last steps must provide the desired product in a form

suitable for final formulation and blending, or for direct shipping. Processes of centrifu-

gation, freeze drying/lyophilisation or organic solvent removal are commonly used.

For antibiotic production, the fermentation broth needs a pretreatment tank to produce crude

and highly purified antibiotic products. The bioprocesses involved in producing antibiotics are

spray or continuously dried crude solids and pure solid in the form of crystalline antibiotic.

Another case of bioprocessing is production of an alternative and renewable fuel from agri-

cultural wastes. Lignocellulosic materials are an abundant and renewable source of energy; in

acid hydrolysis, sugar is the desired product. The acid can be neutralised by adding sodium

hydroxide or any bases; the sugar may not be fermentable in the presence of salts. Solvent

extraction is normally performed to recover acid and reuse/recycle acid for hydrolysis

process. Once fermentable sugar is obtained, it is useful to carry on fermentation for ethanol,

organic acid, enzyme or antibiotic production. Figures 7.2 and 7.3 show the common

Acid

NaOH

Sugar

Unconverted ash

Acid

Raw

material

Hydrolysis

Neutralization

+ ash

FIG. 7.2. Acid recovery by liquid–liquid extraction.

Single-stage extraction

Hydrolysate

organic

solvent

HCl + MIBK

Solvent (amyl-acetate)

Clear solution

Solvent +

amylase

Amylase

bacteria

Bacterial

sludge

Centrifuge

Extraction

FIG. 7.3. Stages of unit operation for product recovery.

Ch007.qxd 10/27/2006 10:44 AM Page 172

DOWNSTREAM PROCESSING 173

separation and extraction processes that are involved in purification of enzyme and fermentable

sugar for related bioprocesses.

When the fermentation broth enters the downstream units, it has high viscosity and needs pre-

treatment. Addition of chemicals and coagulating aids for cell flocculation such as polyelec-

trolytes, CaCl

and colloidal clay are useful pretreatments to recover product downstream.

Settling solids are often used in large-scale waste treatment processes as well as traditional

fermentation industries. Generally, simple centrifugation produces a cell-concentrated stream

of 15% w/v. Also, filtration produces more concentrated cell sludge of up to 20–35% w/v.

7.3 FILTRATION

In filtration, solid particles are separated from a fluid–solid mixture by forcing the fluid

through a filter medium or filter cloth, which retains the solid particles. As a result, solids are

retained by filter media and the filtrate is obtained, which is a clear solution without any solid

particles. The solid particles deposited on the filter form a layer, which is known as filter cake.

The deposited solids create resistance which reduces filter flux. The depth of the filter cake

gradually increases as more solids are retained. The filter cake may create more resistance to

further filtration. Filtration can be performed using either vacuum or positive-pressure equip-

ment. The exerting differential pressure across the filter separates fluid from solid and is

called the filtration pressure drop. Ease of filtration depends on particle properties and fluid

filtrates. The compaction of particles, either soft or hard, compressible or non-compressible,

and the viscosity of the fluid may create different resistances. Fermentation broths are trouble-

some and hard to filter, because of the non-Newtonian behaviour of the broth. Most microbial

filter cakes are compressible. When the filter pores are clogged by cell bodies, then the high

pressure drop results in major problems in the filtration of fermentation broth. Then more

pressure is created and gradually filtration rate drops.

Filter aids are widely used in the fermentation industry to improve the efficiency of filtra-

tion. It is a pre-coated filter medium to prevent blockage or blinding of the filter by solids,

which would otherwise wedge themselves into the pores of the cloth. Filter aid can be added

to the fermentation broth to increase the porosity of the cake as it formed. This is only rec-

ommended when fermentation product is extracellular. Filter aid adds to the cost of filtration.

The minimum quantity needed to achieve the desired result must be established experimen-

tally. Fermentation broths can be pretreated to improve filtration characteristics. Heating to

denature proteins enhances the filterability of mycelial broths such as in penicillin produc-

tion. Alternatively, electrolytes may be added to promote coagulation of colloids into larger,

denser particles, which are easier to filter. The filtration process is affected by the viscosity

and composition of the broth, and the cell cake.

Plate filters are suitable for filtration of batch fermentation broth; accumulated biomass

must be cleaned periodically. A rotary drum vacuum filter is used for a continuous system.

Lignocellulosic material Acid Sugar Solid re

Hydrolysis

æÆæææ ssidues

Ch007.qxd 10/27/2006 10:44 AM Page 173