Bioprocess Principles

7/17/2019 Bioprocess Principles

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BIOPROCESS PRINCIPLES

Biotechnology is a field of applied biology that involves the use of living organisms and

bioprocesses in Engineering, technology, medicine and other fields requiring bioproducts.

Biotechnology also utilizes these products for manufacturing purpose. Modern use of similar

terms includes genetic engineering as well as cell and tissue culture technologies. The concept

encompasses a wide range of procedures for modifying living organisms according to human

purposes — going back to domestication of animals, cultivation of plants, and "improvements"

to these through breeding programs that employ artificial selection and hybridization.

Bioprocess Engineering is a specialization of Boitechnology, Chemical engineering and

Agricultural Engineering. It deals with the design and development of equipment and processes

for the manufacturing of products such as food as feed, pharmaceuticals, chemicals

polymers and paper from biological materials.Bioprocees engineering is a combination of

mathematics, biology and industrial design, and consists of various spectrums like designing

of Fermentors, study of fermentors (mode of operations etc.).It also deals with studying various

biotechnological processes used in industries for large scale production of biological product for

optimization of yield in the end product and the quality of end product. Bio process engineering

may include the work of mechanical, electrical and industrial engineers to apply principles of

their disciplines to processes based on using living cells or sub component of such cells

Bio processing is an essential part of many food, chemical and pharmaceutical industries.

Bioprocess operations make use of microbial, animal and plant cells and components of cells

such as enzymes to manufacture new products and destroy harmful wastes. Use of

microorganisms to transform biological materials for production of fermented foods has its

origins in antiquity. Since then, bioprocesses have been developed for an enormous range of

commercial products, from relatively cheap materials such as industrial alcohol and organicsolvents, to expensive specialty chemicals such as antibiotics, therapeutic proteins and vaccines.

Industrially-useful enzymes and living cells such as bakers‘ and brewers‘ yeast are also

commercial products of bio processing. Our ability to harness the capabilities of cells and

enzymes has been closely related to advancements in microbiology, biochemistry and cell

physiology. Knowledge in these areas is expanding rapidly; tools of modern biotechnology such



as recombinant DNA, gene probes, cell fusion and tissue culture offer new opportunities to

develop novel products or improve Bio processing methods. Visions of sophisticated medicines,

cultured human tissues and organs, biochips for new-age computers, environmentally-compatible

pesticides and powerful pollution-degrading microbes herald a revolution in the role of biology

in industry. Biological systems can be complex and difficult to control; nevertheless, they obey

the laws of chemistry and physics and are therefore amenable to engineering analysis.

Engineering aspects are required in bioprocessing, including design and operation of

bioreactors, sterilisers and product-recovery equipment, development of systems for process

automation and control, and efficient and safe layout of fermentation factories.

Steps in Bioprocess Development

Manufacture of a new recombinant-DNA-derived product such as insulin, growth hormone or

interferon is shown above

Step-1-11 : Genetic Manipulation of the host organism where a gene from animal DNA is

cloned into Escherichia coil. Tools :Petri dishes, micropipettes, micro centrifuges, nano-or

microgram quantities of restriction enzymes, and electrophoresis gels for DNA and protein



fractionation.In terms of bioprocess development, parameters of major importance are stability

of the constructed strains and level of expression of the desired product.

Step-12 : After cloning, the growth and production characteristics of the cells must be measured

as a function of culture environment .Small-scale culture is mostly carried out using shake flasks

of 250-ml to 1-1itre capacity. Medium composition, pH, temperature and other environmental

conditions allowing optimal growth and productivity are determined. Calculated parameters such

as cell growth rate, specific productivity and product yield are used to describe performance of

the organism

Step-13 : Scale-up of the process

The first stage may be a 1- or 2-1itre bench-top bioreactor equipped with instruments for

measuring and adjusting temperature, pH, dissolved-oxygen concentration, stirrer speed and

other process variables.Cultures can be more closely monitored in bioreactors than in shake

flasks so better control over the process is possible. Information is collected about the oxygen

requirements of the cells, their shear sensitivity, foaming characteristics and other

parameters.The viability of the process as a commercial venture is of great interest; information

about activity of the cells is used in further calculations to determine economic feasibility

Step 14 :scaled up again to a pilot-scale bioreactor

Engineers trained in bio processing are normally involved in pilot-scale operations. A vessel of

capacity 100-1000 litres is built according to specifications determined from the bench-scale

prototype.Changing the size of the equipment seems relatively trivial; however, loss or variation

of performance often occurs. Even though the geometry of the reactor, method of aeration and

mixing, impeller design and other features may be similar in small and large fermenters, the

effect on activity of cells can be great. Loss of productivity following scale-up may or may not

be recovered; economic projections often need to be re-assessed as a result of pilot-scale

findings.



Step 15 : Design of the industrial-scale operation

This part of process development is clearly in the territory of bioprocess engineering. As well as

the reactor itself, all of the auxiliary service facilities must be designed and tested. These include

air supply and sterilisation equipment, steam generator and supply lines, medium preparation and

sterilisation facilities, cooling-water supply and process-control network. Particular attention is

required to ensure the fermentation can be carried out aseptically, design of the process must also

reflect containment and safety requirements

Step :16 : Product Recovery (downstream processing)

After leaving the fermenter, raw broth is treated in a series of steps to produce the final product.

Product recovery is often difficult and expensive; for some recombinant - DNA-derived

products, purification accounts for 80-90% of the total processing cost.Downstream processing

depend on the nature of the product and the broth; physical, chemical or biological methods may

be employed.Commercial procedures include filtration, centrifugation and flotation for

separation of cells from the liquid, mechanical disruption of the cells product is intracellular,

solvent extraction, chromatography, membrane filtration, adsorption, crystallisation and

drying.Scientists trained in chemistry, biochemistry, chemical engineering and industrial

chemistry play important roles in designing product recovery and purification "systems.

Step 17 : Packing and Marketing

For new pharmaceuticals such as recombinant human growth hormone or insulin, medical and

clinical trials are required to test the efficacy of the product. Animals are used first, then humans.

Only after these trials are carried out and the safety of the product established can it be released

for general health-care application. Other tests are required for food products. Manufacturing

standards must be met; this is particularly the case for recombinant products where a greater

number of safety and precautionary measures is required.



Bioprocesses treat raw materials and generate useful products. Individual operations or steps

within the process that change or separate components are called unit operations. Although the

specific objectives of bioprocesses vary from factory to factory, each processing scheme can be

viewed as a series of component operations which appear again and again in different systems.

For example, most bioprocesses involve one or more of the following unit operations:

centrifugation, chromatography, cooling, crystallisation, dialysis, distillation, drying,

evaporation, filtration, heating, humidification, membrane separation, milling, mixing,

precipitation, solids handling, solvent extraction. In a typical fermentation process, raw materials

are altered most significantly by reactions occurring in the fermenter. However physical changes

before and after fermentation are also important to prepare the substrates for reaction and to

extract and purify the desired product from the culture broth .Fermentation broths are complex

mixtures of components containing products in dilute solution. In bioprocessing, any treatment

of the culture broth after fermentation is known as downstream processing. The purpose of

downstream processing is to concentrate and purify the product for sale; in most cases this

requires only physical modification.

Recovery scheme will be different, downstream processing follows a general sequence of

steps.

Cell removal. A common first step in product recovery is removal of cells from the fermentation

liquor. This is necessary if the biomass itself is the desired product. e.g. bakers' yeast, or if the

product is contained within the cells. Removal of cells can also assist recovery of product from

the liquid phase. Filtration and centrifugation are typical unit operations for cell removal.

Primary isolation. A wide variety of techniques is available for primary isolation of

fermentation products from cells or cell-free broth. The method used'depends on the physical and

chemical properties of the product and surrounding material. Typically, processes for primary

isolation treat large volumes of material and are relatively non-selective; however significant

increases in product quality and concentration can be accomplished. Unit operations such as

adsorption, liquid extraction and precipitation are used for primary isolation



Purification. Processes for purification are highly selective and separate the product from

impurities with similar properties. Typical unit operations are chromatography, ultrafiltration and

fractional precipitation.

Final isolation. The final purity required depends on the product application. Crystallisation,

followed by centrifugation or filtration and drying, are typical operations used for high-quality

products such as pharmaceuticals.

Process Flow Diagrams

Because of the complexity of large-scale manufacturing processes, communicating information

about these systems requires special methods. Flow diagrams or flow sheets are simplified

pictorial representations of processes and are used to present relevant process information and

data. Flow sheets vary in complexity from simple block diagrams to highly complex schematic

drawings showing main and auxiliary process equipment such as pipes, valves, pumps and by-

pass loops.

Production of the antibiotic, bacitracin



Discovery of Penicillin

Penicillin was discovered by chance, after Alexander Fleming accidentally left a dish of

staphylococcus bacteria uncovered for a few days. He returned to find the dish dotted with

bacterial growth, apart from one area where a patch of mould (Penicillin notatum) was growing.

The mould produced a substance, named penicillin by Fleming, which inhibited bacterial growth

and was later found to be effective against a wide range of harmful bacteria. However, it was not

until World War II that penicillin, the first antibiotic, was finally isolated by Howard Florey and

Ernst Chain. Fleming, Florey and Chain received a Nobel prize in 1945, for their discovery

which revolutionised medicine and led to the development of life saving antibiotics.

Manufacture of Penicillin

Though the precise and exact compositions of the penicillin-production media really employed in

any industry are more or less impossible to quote and determine, by virtue of the fact that such

information(s) are regarded to be the 'trade secrets' or patented by the actual users. Nevertheless,

a large segment of these commonly used media invariably comprises of such ingredients as: corn

steepliquor solids, lactose, glucose, calcium carbonate, potassium di hydrogen phosphate

[KH2PO4 ], edibleoil, and a penicillin precursor. Jackson (1958) promulgated a very useful and

typical medium having essentially the following composition

(1) The pH after sterilization is carefully maintained between 5.5 to 6.0.

(2) Higher lactose content ranging between 4 to 5% is desired with vigorously increased aeration

and agitation environments maintained within the fermentor (i.e., bioreactor).

(3) The 'production media' contains both 'lactose' and 'precursor' which are not included in the

inoculums media



Penicillin Production

1.The completed penicillin fermentation culture is subjected to filtration by the help of

heavy duty rotary vacuum filter to get rid to the mycelium plus other unwanted solid residues.

2. The pH of the clear filtered fermented broth is carefully brought down between 2 to 2.5 by the

addition of a calculated amount of either phosphoric or sulphuric acid so as to convert the

resulting penicillin to its anionic form.

3. The resulting fermented broth (pH 2-2.5) is extracted immediately by using countercurrent

solvent extractor, with an appropriate organic solvent e.g., amyl acetate, butylacetate, or methyl

isobutyl ketone.

4. Penicillin, thus obtained, is back extracted into aqueous medium from the corresponding

organic solvent by the careful addition of requiste quantum of KOH or Na0H to give rise to the

formation of the corresponding potassium or sodium salt of the penicillin.

5. The resulting aqueous solution, containing the respective salt of penicillin, is again acidified

and re-extracted with the organic solvent methyl isobutyl ketone

6. The resulting solvent extract is finally subjected to a meticulous back-extraction with aqueous

NaOH preferably, a number of times till extraction of penicillin is completed; and from this



combine of aqueous extractions different established procedures are adopted to afford the

penicillin to crystallize out either as sodium or potassium penicillin

7. The crystalline penicillin thus obtained is washed, dried under vacuum, and the final product

must conform to the requirements/specifications

Ethanol Production

Ethanol has been made since ancient times by the fermentation of sugars. All beverage ethanol

and more than half of industrial ethanol is still made by this process. Simple sugars are the raw

material. Zymase, an enzyme from yeast, changes the simple sugars into ethanol and carbon

dioxide. The fermentation reaction, represented by the simple equation

C6H12O6 2 CH3CH2OH + 2 CO2

It is actually very complex, and impure cultures of yeast produce varying amounts of other

substances, including glycerine and various organic acids. In the production of beverages, such

as whiskey and brandy, the impurities supply the flavor. Starches from potatoes, corn, wheat,

and other plants can also be used in the production of ethanol by fermentation. However, the

starches must first be broken down into simple sugars. An enzyme released by germinating



barley, diastase, converts starches into sugars.Thus, the germination of barley, called malting, is

the first step in brewing beer from starchy plants, such as corn and wheat.

The ethanol produced by fermentation ranges in concentration from a few percent up to about 14

percent. Above about 14 percent, ethanol destroys the zymase enzyme and fermentation stops.

Ethanol is normally concentrated by distillation of aqueous solutions, but the composition of the

vapor from aqueous ethanol is 96 percent ethanol and 4 percent water. Therefore, pure ethanol

cannot be obtained by distillation. Commercial ethanol contains 95 percent by volume of ethanol

and 5 percent of water. Dehydrating agents can be used to remove the remaining water and

produce absolute ethanol.

Manufacture of Lactic Acid

Lactic acid has been first introduced to us as early as 1780 as a sour component of milk. Ever

since we have found its applications in food, pharmaceutical, cosmetic industries etc.

Lactic acid bacteria compose a group of bacteria that degrade carbohydrate with the production

of lactic acid. Examples of genera that contain lactic acid bacteria

include Streptococcus, Lactobacillus, Lactococcus, and Leuconostoc



Enzyme Production

Sources of Industrial Enzymes can be

a. Plant

b. Animals

c. Microorganism

Selection of industrial enzymes from any of these sources depends on the following factors:

a. specificity

b. pH

c. Thermo stability

d. activation or inhibition

e. availability and cost

Enzymes and their producer microorganisms



Most industrial enzymes are products of batch processes and few are currently pro-duced via

continuous fermentation. The fermenters for bulk enzyme production are up to 100m3capacity,

but fine enzymes may be produced on smaller scales of a few hundred litres or less. Most

fermenters are stirred tank reactors that are operated under aseptic conditions and use low cost

undefined complex media.

As in the development of any fermentation process, enzyme production processes includes the

following steps

• search for a suitable producer organism.

• screening of microorganism and selection to determine enzyme properties, such as opti-

mum pH and heat resistance, and examination of the ability to secrete the target enzyme.

• The fermentation system and ferementation media to be determined. Conditions for

maximum production of the enzyme per unit of biomass, using inexpensive car-bon and

nitrogen feedstocks, must be identified.

• Downstream processing involves separation,. Purification, stabilization and preservation

Enzyme stability has to be determined as it can influence the timing, and operations used

in, downstream processing.



• The level of purification applied varies considerably depend-ing on whether the enzyme

is intracellular or extracellu-lar, and on its end use.

Generalised manufacture of enzymes



Processing of Biological Materi

Filtration

In filtration, solid particles are s

a filter medium or filter cloth wh

the deposit or filter cake increas

performed using either vacuum

across the filter to separate

Fermentation broths can be diff

the cells and the viscous non-N

compressible, i.e. the porosity o

This can be a major problem cau

al

parated from a fluid-solid mixture by forcing

ich retains the particles. Solids are deposited o

s in depth, pose a resistance to further filtration

or positive-pressure equipment. The pressure d

luid from the solids is called the filtratio

cult to filter because of the small size and gel

wtonian behaviour of the broth. Most microbi

f the cake declines as pressure drop across th

sing reduced filtration rates and greater loss of

the fluid through

the filter and, as

.Filtration can be

ifference exerted

pressure drop.

atinous nature of

l filter cakes are

filter increases.

roduct.



Filter Aids:

Filter aids such as diatomaceous earth have found widespread use in the fermentation industry to

improve the efficiency offiltration.Filter aids are applied in two ways. As shown in Figure, filter

aid can be used as a pre-coat on the 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 also

be added to the fermentation broth to increase the porosity of the cake as it forms.

Centrifugation

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

is desired. In bioprocessing, centrifugation is used to remove cells from fermentation broth, to

eliminate cell debris, to collect precipitates, and to prepare fermentation media such as in

clarification of molasses or production of wort for brewing.Centrifugation is most effective when

the particles to be separated are large, the liquid viscosity is low, and the density difference

between particles and fluid is great. It is also assisted by large centrifuge radius and high

rotational speed.In centrifugation of biological solids such as cells, the particles are very small,

the viscosity of the medium can be relatively high, and the particle density is very similar to the

suspending fluid.



Disc-stack bowl centrifuge

Cell Disruption

For products such as enzymes and recombinant proteins which remain in the biomass, cell

disruption must be carried out to release the desired material.Variety of methods is available to

disrupt cells. Mechanical options include grinding with abrasives, high-speed agitation, high-

pressure pumping and ultrasound. Non-mechanical methods such as osmotic shock, freezing and

thawing, enzymatic digestion of cell walls, and treatment with solvents and detergents can also

be applied.

High-pressure pump incorporates an adjustable valve with restricted orifice through which cells

are forced at pressures up to 550 atm. The homogeniser is of general applicability for cell

disruption, although the homogenising valve can become blocked when used with highly

filamentous organisms.



Aqueous Two-Phase Liquid Extraction

In liquid extraction of fermentation products, components dissolved in liquid are recovered by

transfer into an appropriate solvent. Extraction of penicillin from aqueous broth using solvents

such as butyl acetate, amyl acetate or methyl isobutyl ketone, and isolation of erythromycin

using pentyl or amyl acetate are examples.Solvent extraction techniques are also applied for

recovery of steroids, purification of vitamin B12 from microbial sources, and isolation of

alkaloids such as morphine and codeine from raw plant material.

Techniques are being developed for aqueous two-phase extraction of these molecules. Aqueous

solvents which form two distinct phases provide favourable conditions for separation of proteins,

cell fragments and organelles with protection of their biological activity.

Chromatography

Chromatography is a separation procedure for resolving mixtures and isolating components. The

basis of chromatography is differential migration, i.e. the selective retardation of solute

molecules during passage through a bed of resin particles.



As solvent flows through the column, the solutes travel at different speeds depending on their

relative affinities for the resin particles. As a result, they will be separated and appear for

collection at the end of the column at different times. The pattern of solute peaks emerging from

a chromatography column is called a chromatogram.The fluid carrying solutes through the

column or used for elution is known as the mobile phase; the material which stays inside the

column and effects the separation is called the stationary phase.Chromatography is a high-

resolution technique and therefore suitable for recovery of high-purity therapeutics and

pharmaceuticals. Chromatographic methods available for purification of proteins, peptides,

amino acids, nucleic acids, alkaloids, vitamins, steroids and many other biological materials

include adsorption chromatography, partition chromatography, ion-exchange chromatography,

gel chromatography and affinity chromatography. These methods differ in the principal

mechanism by which molecules are retarded in the chromatography column.

Distillation

Distillation is simply defined as a process in which a liquid or vapor mixture of two or more

substances is separated into its component fractions of desired purity, by the application and

removal of heat.



Evaporation

Evaporation is the removal of aevaporates and solids do not eva

increased.

Tie substance balance.

Any material which does not ge

substance balance the quantities

Crystallization

In process industries products ar

different unit operations such

Crystallization is carried out wh

component. This is done by sup

namely cooling, evaporation an

larger extent with increase in te

becomes super saturated by sim

of crystallization, adiabatic c

simultaneously carried out with

from solid mixture. The purifica

portion of solvent by boiling the solution. Oporate. By evaporation the solid concentration

t altered in a process is called a tie substance.

of the streams are evaluated.

e produced with impurities in the reactors. To

as distillation, evaporation, crystallization

n a solid product is to be recovered from its s

er saturating the solution by one or more of t

adiabatic cooling. If the solubility of the s

mperature (strong function of temperature) a s

ply cooling and temperature reduction.To incr

ooling is normally carried out (evaporati

out the addition of heat). Some times a solid h

tion of the mixture (separation of one compon

ly liquid portionin the solution is

By writing a tie

et pure products

etc. are used.

lution with other

e three methods

lute increases to

aturated solution

ase the capacity

n and cooling

s to be removed

nt) is carried out



by treating the solid mixture with a suitable solvent so as to dissolve only the desired component

and then crystallizing it. The solubility of solute plays an important role in the crystallization

operation

Drying

Drying refers to an operation in which the moisture of a substance is removed by thermal means

the removal of relatively small amount of water or other liquids from the solid material. Drying

is one of the oldest methods of preserving food. Primitive societies practiced the drying of meat

and fish in the sun long before recorded history.

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Bioprocess Principles