Ghasem D. Najafpour. Biochemical Engineering and Biotechnology

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344 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY

microorganisms. The medium from a make-up vessel flowing through the exchanger is held

in the coil, and then goes through the heat exchanger, heating more unsterile medium while

become cool itself, as it is collected in a sterile fermenter. Figure 15.2 presents the sequence

of piping media goes through, along with steam. Finally, the media is collected in a vacuum

flash drum.

Heat economisers are important for large-scale plant unit in continuous sterilisation. For

a small-scale bioreactor use of direct steam injection is much simpler to operate. A heat

exchanger is then needed with cooling water to bring the medium back to ambient tempera-

ture. Therefore jacketed vessels are commonly used with cooling water. There are advan-

tages and disadvantages to batch and continuous sterilisation. The energy savings are

related to the use of an economiser and direct stream or indirect sterilisation. In a large-

scale system economy and role decide which one is the most suitable process to be imple-

mented. For each case the specific design needs to be evaluated in terms of the thermal

efficiency and fixed costs involved.

15.4 HOT PLATES

In directing steam heating, continuous sterilisation is preferable. The medium passed

through a preheater may reach about 90 ⬚C; then quite fast sterilisation may take at 140 ⬚C.

Figure 15.3 shows a counter-current hot-plate heat exchanger is normally used for contin-

uous sterilisation. Hot-plate heat exchangers are extremely efficient and easily maintained

if any fouling or scale deposition takes place. Normally fouling is serious problem, occur-

ing by deposition of proteins on the hot surface of the exchanger, which causes reduction

in the overall heat transfer coefficient. Plate exchangers are easily separated and cleaned.

Special care must be taken when the media contains any agglomerated particles. Correla-

tion is required to calculate the suitable residence time for sterilisation. The choice of a

Medium

Steam injector

Holding section

Expansion valve

Sterile medium

Vacuum

flash

chamber

FIG. 15.2. Continuous sterilisation.

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STERILISATION 345

suitable sterilisation process is based on economics and cost of the heat exchangers to

reduce energy consumption. In a suitable exchanger, the larger the heat transfer coefficient,

the greater the energy recovery.

15.5 HIGH TEMPERATURE STERILISATION

Fast sterilisation is performed at high temperature. High-temperature sterilisation requires

short holding times. This technique is used in the fast preparation of nutrient media for

industrial bioprocesses and in pasteurising milk. A short retention time may favour media

with heat-sensitive proteins and cause less damage to the biochemical composition of the

media than more prolonged times at lower temperatures. The ability of high temperatures

to perform rapid sterilisation is related to activation energies. That is affected by how fast

bacteria are killed in an elevated temperature. On the other hand, long sterilisation at high

temperatures may destroy the protein and biochemical composition of the media. Short-

duration sterilisation at high-temperatures are more lethal to organisms and less chemically

damaging than are longer sterilisation processes at lower temperatures. Sterilisation at high

temperature is recommended for 3 minutes at 134 ⬚C. It is preferable to 20 minutes at 115 ⬚C

in conventional operation.

15.6 STERILISED MEDIA FOR MICROBIOLOGY

Sterile media are used for pure culture. The media used to culture microorganisms depend

on the living conditions of the microorganisms. Media compositions must be identified

Hot liquid out

Cold liquid in

Cold liquid out

Hot liquid in

FIG. 15.3. Counter current hot plate heat exchanger.

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346 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY

based on the needs of the organisms for carbon, nitrogen and phosphorus. Nutrients such

as protein and sugars are added at wide ranges of pH. The acidic, neutral and alkaline con-

ditions are defined based on differentiation of microbes in their biochemical reactions. Colour

indicators are used to observe any transition of pH. Buffer is used in the media to maintain

constant pH. Growth factors and growth stimulants are also used to accelerate microbial

growth and to maintain exponential growth. The most common media in the liquid phase

is nutrient broth; in the solid phase, nutrient agar is used to propagate microorganisms.

When microbiological media is prepared, it has to be sterilised before any inoculation of

organisms. Sterilisation is used to eliminate any microbial contamination that may originate

from air, glassware or hands. Microbes propagate quite quickely: within a few hours there

will be thousands of organisms reproduced in the media. To control the growth of unwanted

organisms, the media has to be sterilised quickly before any microbes start to utilise the

nutrients. The sterilisation process makes the media absolutely free of contaminants and

guarantees that the medium will stay sterile, so that is used only for the desired organisms.

The kinetics of culture media sterilisation describe the rate of destruction of microor-

ganisms by steam using a first-order chemical reaction rate model. As the population of

microorganisms (N) decreases with time, the rate is defined by the following equation:

(15.6.1)

where N is the number of viable organisms present in the culture media, t is the retention

time or sterilisation time, and k

is the reaction rate constant as it is known for a specific

death rate. Using separation of variables and integrating with initial condition, the follow-

ing useful expression is obtained:

(15.6.2)

where N

is the number of viable organisms present in the media at the starting point before

sterilisation. Now take the natural log of (15.6.2); it is reduced to a linear model:

(15.6.3)

The graphical presentation of the equation shows a straight line with a negative slope for

. As the death rate constant follows Arrhenius’ law,

the death rate constant is tempera-

ture dependent. The value of k

is about 0.02 min

⫺1

at 100 ⬚C, the death rate constant

increases by 10-fold at 110 ⬚C and 100-fold at 120 ⬚C.

where k

is the death rate constant at a reference temperature also known as Arrhenius’

constant, R is the gas constant, T is the absolute temperature, E is the activation energy,

ERT

e⫽

⫺ /

()

Nt N

()⫽

⫺

⫽

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STERILISATION 347

which is 60–70 kcal per mole for microorganisms, 100–150 kcal per mole for spores, and

30–40 kcal per mole for media with vitamins and protein solution.

Figure 15.4 shows the linear model for (15.6.3), the loss of cell viability at various tem-

peratures. As the temperature increases from 105 to 121 ⬚C, the value for the slope of the

line increases. This means that the number of viable cells at a fixed time of sterilisation will

drastically decrease as the temperature increases by 16 ⬚C.

15.6.1 Sterilisation of Media for Stoke Cultures

To clear slant tubes, culture agar solution is prepared. To have clear agar solution, it has to

boil. Agar is a polysaccharide. It is not soluble in cold water, so heating helps to dissolve it

in water. Once the solution is dispensed in the tube, it goes for sterilisation. Agar is in the

solid state at room temperature, about 35 ⬚C.

15.6.2 Sterilisation of Bacterial Media

Often, sterilisation of culture media is done in an autoclave at temperatures between 121 and

134 ⬚C. It is good to know the damage caused to the medium by heating it. Heating the cul-

ture media, which contains peptides, sugars, vitamins, minerals and metals, results in nutrient

destruction, either thermal degradation or reaction between the components of the media.

Protein in the media is denatured by high temperature, or sugars are easily caramelised.

During the heat treatment, toxic compounds are formed, which may retard or inhibit the

microbial growth. For minimal damage to the ingredients of the media, it is important to opti-

mise and minimise the heating and holding time of the sterilisation process, respectively.

15.6.3 Sterilise Petri Dishes

There are several ways to handle Petri dishes. The standard dishes are 15–20 mm deep and

12–15 cm in diameter. They are normally available in glass and also transparent plastic.

Petri dishes are individually wrapped. The media is separately sterilised while we add the

sterilised media to the Petri dishes in front of a flame. It is recommended to use 20–25 ml

Time, t

tN )(

increasing

-k

= 105 °C

=121 °C

FIG. 15.4. The loss of cell viability at various temperatures.

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348 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY

of medium in each Petri dish. These are all called culture dishes. The media contains about

1.5–2% agar. After the media cools off it solidifies. The Petri dishes with the nutrient agar

should be sterilised and stored in a refridgerator.

It is common to sterilise the media and Petri dishes separately. When the medium is

cooled to about 55 ⬚C, in front of a flame or in a laminar flow chamber, lift the lid of the dish

enough to pour about 25 ml of the medium to the desired depth and lower the lid in place.

It is best to gently move the Petri dish in way that spreads a thin layer of agar uniformly

without any air bubbles. Distribution of media in the Petri dishes should be done in front

of a flame. Most plastic Petri dishes are made of polystyrene and are not autoclaveable.

Plastic Petri dishes are easily deformed during sterilisation at high temperature. Some plastic

dishes can be autoclaved, but they are more expensive. Please follow the instructions given

by the manufacturer or obtain information from catalogues.

It is recommended to autoclave glass dishes and medium separately. If your dishes are

autoclaveable, you may dispense the agar into the dish and then autoclave it. In this case it

is best to cool the dishes in the autoclave or in the pressure cooker to reduce the amount of

water that will condense on the cover of the dish.

15.7 DRY HEAT STERILISATION

Dry heat sterilisation is used for equipment that can withstand high temperature and dry heat

but cannot withstand wet or steam autoclave. This method is often used for glassware as it

dries and sterilises in one operation. The pipets must be wrapped in dustproof aluminum

foil or placed in metal pipette cans. The can lids are removed during heating and replaced

after sterilisation, that is before any dust can get in the can. Disposable items are not recom-

mended for dry heat sterilisation. This method may only be good for permanent reusable glass

pipettes.

Normal laboratory glassware must first be washed and cleaned. It has to be rinsed with

deionised water. The clean glassware is sterilised in an oven set at 200 ⬚C for 1–4 hours. It is

suitable to cover glassware with aluminum foil to maintain aseptic conditions after removing

the glassware from the oven. If aluminum foil is not available, special heat-resistant wrap paper

can be used. The sterile glassware must be protected from the air, which has micro-flora,

or any contaminants. Avoid the use of any plastic caps and papers. Detach any labelling tape

or other flammable materials, as they are fire hazards.

15.8 STERILISATION WITH FILTRATION

Certain media components are susceptible to heat; it is going to be denatured if it is heated.

Therefore they must be added to the media after autoclaving. To do so, it is necessary to carry

out filtration, the components using a 0.22 ␮m pore size filter that is appropriate to the sol-

vent used. Filters are normally available from Whatman, or Fisher Scientific. It is recom-

mended you consult filter experts or suppliers about the solvent and the special application

you are intending to implement for the desired filters for the process. Normally filtration of

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STERILISATION 349

media is used instead of sterilised media with an autoclave. To filter sterilised media, filter

the water using a 0.45 ␮m pore size filter, before using a 0.22 ␮m pore size filter for final

sterilisation.

15.9 MICROWAVE STERILISATION

Rapid sterilisation of media is achieved by using microwave ovens. Most plant tissue culture

media can be sterilised using a microwave, although it may not be suitable with a medium

containing complex additives like oatmeal.

15.10 ELECTRON BEAM STERILISATION

Electron beam sterilisation is a high-voltage potential established between a cathode and an

anode in an evacuated tube. The cathode emits electrons, as a cathodic ray or electron beam.

A high intensity of electrons is produced. These electrons are accelerated to extremely high

velocities. These accelerated electron intensities have great potential as a bacteriocide. Most

electron beams operate in a vacuum. As a result the unwanted organisms in the media vanish

and the media is sterilised.

The electron accelerator equipment producing the high-voltage beam also has various

applications in the medical field and surgical supplies. Electron beam sterilisation is a suc-

cessful technology used for sterilisation of disposable medical appliances and devices with

a wide range of densities. The electron beam inactivates microorganisms that cause destruc-

tion of biomolecules, and results in death of microbes by an indirect chemical reaction.

Irradiation by gamma rays is a similar mechanism. Advanced electronics precisely control

the use of electron beams in the sterilisation of medical devices. Electron beam sterilisation

results in less material degradation than with gamma irradiation. Medical products are ster-

ilised in the original shipping containers, saving lots of time and maintaining integrity of

the original package.

15.11 CHEMICAL STERILISATION

Chemical agents are used to sterilise heat-sensitive equipment. Chemical solutions are used

as a suitable method for sterilising long pipettes and glassware. Normally at pilot scale in

the absence of life, steam and chemical agents are often used. Application of an oxidising

agent such as 10% chlorox for 20 minutes or longer proves the system operates without any

contamination. Excess amounts of chemical agent have to be removed; otherwise organisms

are able to grow in a toxic environment. The bleach (sodium hypochlorite) then needs to

be removed by rinsing with sterile water or rubbing with ethyl alcohol, 70% ethanol–water

(pH ⫽ 2) or an alcohol solution of 70% iso-propanol without recontaminating the glassware

or tools. Ethanol is commonly used for cleaning surfaces. Use of bleach on metal devices is

not suitable as it corrodes metals rapidly. Ethylene oxide in the gas phase is commonly used.

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350 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY

This is a special chemical effectively used for column bioreactors. It is a volatile compound

and strong oxidising agent. It boils at ambient temperature, therefore the solution of ethyl-

ene oxide (liquid phase) must be stored in a refrigerator (4 ⬚C). An excellent oxidising agent

such as a 3% sodium hypochlorite is used for chemical sterilisation of equipment.

REFERENCES

1. Pelczar, M.J., Chan, E.C.S. and Krieg, N.R., “Microbiology”. McGraw-Hill, New York, 1986.

2. Scragg, A.H., “Bioreactors in Biotechnology, A Practical Approach”. Ellis Horwood Series in Biochemistry and

Biotechnology, New York, 1991.

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

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

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

6. Matthews, I.P., Gibson, C. and Samuel, A.H., J Biomed. Mat. Res. 23, 143 (1989).

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CHAPTER 16

Membrane Separation Processes

16.1 INTRODUCTION

Membranes have gained an important place in chemical technology and are used in a broad

range of applications ranging from pharmaceuticals to water treatment. Industrial applications

are divided into six main subgroups: reverse osmosis, nanofiltration, ultrafiltration, microfil-

tration, gas separation, pervaporation and electrodialysis.

The key property that is exploited

is the ability of a membrane to control the permeation rate of a chemical species through the

membrane.

In controlled drug delivery, the goal is to moderate the permeation rate of a drug

from a reservoir to the body. In separation applications, the goal is to allow one component

of a mixture to permeate the membrane freely, while hindering permeation of other com-

ponents.

This chapter provides a general introduction to membrane science and technology.

16.2 TYPES OF MEMBRANE

This chapter is limited to synthetic membranes, excluding all biological structures, but the

topic is still large enough to include a wide variety of membranes that differ in chemical and

physical composition and in the way they operate. In essence, a membrane is nothing more

than a discrete, thin interface that moderates the permeation of chemical species in contact

with it. This interface may be molecularly homogeneous, i.e., completely uniform in compo-

sition and structure; or the interface may be chemically or physically heterogeneous, e.g., con-

taining holes or pores of finite dimensions or consisting of some form of layered structure.

A normal filter meets this definition of a membrane, but, by convention, the term ‘filter’ is

usually limited to structures that separate particulate suspensions larger than 1–10␮m.

3,4

The

351

This case study was contributed by:

Ghasem Najafpour,

Nidal Hilal,

and Abdul Latif Ahmad

Biochemical Engineering, Program Chairman of Biotechnology, Faculty of Chemical Engineering,

Noshirvani Institute of Technology, University of Mazandaran, Babol, Iran.

Reader in Chemical Engineering, Director of Centre for Clean Water Technologies, University of

Nottingham, Deputy Director of Research/School of Chemical, Environmental and Mining Engineering,

University Park, Nottingham, NG7 2NR, United Kingdom.

Membrane Technology, School of Chemical Engineering, Engineering Campus, Universiti Sains

Malaysia, Seri Ampangan, 14300 Nibong Tebal, S.P.S., Pulau Pinang, Malaysia.

Ch016.qxd 10/27/2006 10:52 AM Page 351

principal types of membrane are shown schematically in Figure 16.1 and are described

briefly in the following pages.

16.2.1 Isotropic Membranes

16.2.1.1 Microporous Membranes

A microporous membrane is very similar in structure and function to a conventional filter. It

has a grid, highly voided structure with randomly distributed, interconnected pores. However,

these pores differ from those in a conventional filter by being extremely small, of the order of

0.01–10 ␮m in diameter. All particles larger than the largest pores are completely rejected by

the membrane. According to the pore size distribution of the membrane, the particles smaller

than the largest pores and the particles larger than the smallest pores are partly rejected.

Particles much smaller than the smallest pores are absolutely passed through the membrane.

Thus, separation of solutes by microporous membranes is mainly a function of molecular

size and pore size distribution. In general, only molecules that differ considerably in size can

be separated effectively by microporous membranes, e.g., in ultrafiltration and microfiltra-

tion.

5,6

Isotropic membranes are shown in Figure 16.1a.

16.2.1.2 Non-porous, Dense Membranes

Non-porous, dense membranes consist of a dense film through which permeats are trans-

ported by diffusion under the driving force of a pressure, concentration or electrical potential

gradient. The separation of various components of a mixture is related directly to their relative

transport rates within the membrane, which are determined by their diffusivity and solubility in

the membrane material. Thus, non-porous, dense membranes can separate permeats of similar

352 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY

(a)

(b)

Isotropic membranes

Anisotropic membranes

Isotropic microprous

membrane

Non-porous dense

membrane

Supported liquid

membrane

Liquid-filled

pores

Polymer

matrix

Thin-film composite

anisotropic membranes

Loeb−Sourirajan

anisotropic membranes

Electrically charged

membrane

coo

FIG. 16.1. Schematic diagrams of the principal types of membrane.

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size if their concentration in the membrane material (i.e. their solubility) differs significantly.

Most gas separation, pervaporation and reverse osmosis membranes use dense membranes

to perform the separation. Usually these membranes have an anisotropic structure to

improve the flux.

16.2.1.3 Electrically Charged Membranes

Electrically charged membranes are dense or microporous. Most commonly these mem-

branes are very fine microporous, with the pore walls carrying fixed positively or negatively

charged ions. A membrane with fixed positively charged ions is referred to as an anion-

membrane because it binds anions in the surrounding fluid. Similarly, a membrane contain-

ing fixed negatively charged ions is called a cation-exchange membrane. Separation with

charged membranes is achieved mainly by exclusion of ions of the same charge as the fixed

ions of the membrane structure, and to a much lesser extent by the pore size. The separa-

tion is affected by the charge and concentration of the ions in solution. For example, mono-

valent ions are excluded less effectively than divalent ions, and in solutions of high ionic

strength, selectivity decreases. Electrically charged membranes are used for processing

electrolyte solutions in electrodialysis.

7,8

16.2.2 Anisotropic Membranes

The transport rate of a species through a membrane is inversely proportional to the membrane

thickness. High transport rates are desirable in membrane separation processes for economic

reasons; therefore, the membrane should be as thin as possible. Conventional film fabrica-

tion technology limits manufacture of mechanically strong, defect-free films to about 20 ␮m

thickness.

2,9

The development of novel membrane fabrication techniques to produce

anisotropic membrane structures has been one of the major breakthroughs of membrane

technology during the past 30 years. Anisotropic membranes consist of an extremely thin

surface layer supported on a much thicker, porous structure. The surface layer and its struc-

ture may be formed in a single operation or separately. In composite membranes, the layers

are usually made from different polymers. The separation properties of permeation rates of

the membrane are determined exclusively by the surface layer; the substructure functions as

a mechanical support. The advantages of the higher fluxes provided by anisotropic mem-

branes are so great that almost all commercial processes use such membranes.

16.2.3 Ceramic, Metal and Liquid Membranes

The discussion so far implies that membrane materials are organic polymers, and in fact

most membranes used commercially are polymer-based. However, in recent years, interest

in membranes made of less conventional materials has increased. Ceramic membranes, a

special class of microporous membranes, are being used in ultrafiltration and microfiltra-

tion applications for which solvent resistance and thermal stability are required. Dense, metal

membranes, particularly palladium membranes, are being considered for the separation of

hydrogen from gas mixtures, and supported liquid films are being developed for carrier-

facilitated transport processes.

MEMBRANE SEPARATION PROCESSES 353

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