FE 462 BIOCHEMICAL ENGINEERING

Agitation and Aeration
Introduction
BASIC MASS-TRANSFER CONCEPTS
The path of gaseous substrate from a gas bubble to an organelle in a microorganism can be divided into several steps as follows:

1. Transfer from bulk gas in a bubble to a relatively unmixed gas layer

2. Diffusion through the relatively unmixed gas layer

Molecular Diffusion in Liquids
When the concentration of a component varies from one point to another, the component has a tendency to flow in the direction that will reduce the local differences in concentration.
Molar flux of a component A relative to the average molal velocity of all constituent JA is proportional to the concentration gradient dCA/dz as
Diffusivity
The kinetic theory of liquids is much less advanced than that of gases. Therefore, the correlation for diffusivities in liquids is not as reliable as that for gases. Among several correlations reported, the Wilke-Chang correlation (Wilke and Chang, 1955) is the most widely used for dilute solutions of nonelectrolytes,
Example
Solution:

Equation 1 suggests that the quantity DABμ/T is constant for a given liquid system. Though this is an approximation, we may use it here to estimate the diffusivity at 40°C. Since the viscosity of water at 40°C is 6.529 X 10-4 kg/m s from the handbook,

Gas Sparging
Air under pressure is supplied through a tube end consists of an ‘O’ ring with very fine holes or orifices. The size of bubbles depends on the size of hole and type of sparger. For very fine bubbles with effective gas dispersion, a micro-sparger is used in the fermenter.
A micro-sparger is in fact a highly porous ceramic material and is used instead of a gas sparger. The size of bubbles affects the mass transfer process. Smaller bubble size provides more surface area for gas exposure, so a better oxygen transfer rate is obtained.
Gas Hold-up
Gas hold-up is one of the most important parameters characterizing the hydrodynamics in a fermenter. Gas hold-up depends mainly on the superficial gas velocity and the power consumption, and often is very sensitive to the physical properties of the liquid. Gas hold-up can be determined easily by measuring level of the aerated liquid during operation ZF and that of clear liquid ZL. Thus, the average fractional gas hold-up H is given as;
Power Consumption
Figure shows Power number-Reynolds number correlation in an agitator with four baffles (Rushton et al., 1950) for three different types of impellers. The power number decreases with an increase of the Reynolds number and reaches a constant value when the Reynolds number is larger than 10,000. At this point, the power number is independent of the Reynolds number.

The power required by an impeller in a gas sparged system Pm is usually less than the power required by the impeller operating at the same speed in a gas-free liquids Pmo. The Pm for the fIat-blade disk turbine can be calculated from Pmo (Nagata, 1975), as follows:

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FE 462 BIOCHEMICAL ENGINEERING

Sterilization
INTRODUCTION
Most industrial fermentations are carried out as pure cultures in which only selected strains are allowed to grow. If foreign microorganisms exist in the medium or any parts of the equipment, the production organisms have to compete with the contaminants for the limited nutrients. The foreign microorganisms can produce harmful products which can limit the growth of the production organisms. Therefore, before starting fermentation, the medium and all fermentation equipment have to be free from any living organisms, in other words, they have to be completely sterilized. Furthermore, the aseptic condition has to be maintained.
STERILIZATION METHODS
Sterilization of fermentation media or equipment can be accomplished by destroying all living organisms by means of heat (moist or dry), chemical agents, radiation (ultraviolet or X-rays), and mechanical means (some or ultrasonic vibrations). Another approach is to remove the living organisms by means of filtration or high-speed centrifugation.
Heat is the most widely used means of sterilization, which can be employed for both liquid medium and heatable solid objects. It can be applied as dry or moist heat (steam).
Laboratory autoclaves are commonly operated at a steam pressure of about 30 psia, which corresponds to 121°C. Even bacterial spores are rapidly killed at 121 °C.
Many cellular materials absorb ultraviolet light, leading to DNA damage and consequently to cell death. Wavelengths around 265 nm have the highest bactericidal efficiency.
Sonic or ultrasonic waves of sufficient intensity can disrupt and kill cells.
Filtration is most effectively employed for the removal of microorganisms from air or other gases.
Chemical agents can be used to kill microorganisms as the result of their oxidizing or alkylating abilities. However, they cannot be used for the sterilization of medium because the residual chemical can inhibit thefermentation organisms.
THERMAL DEATH KINETICS
Thermal death of microorganisms at a particular temperature can be described by first-order kinetics:
which shows the exponential decay of the cell population. The temperature dependence of the specific death rate kd can be assumed to follow the Arrhenius equation:
DESIGN CRITERION
From above equations, the design criterion for sterilization Ñ can be defined as
Batch Sterilization
Sterilization of the medium in a fermenter can be carried out in batch mode by direct steam sparging, by electrical heaters, or by circulating constant pressure condensing steam through heating coil. The sterilization cycles are composed of heating, holding, and cooling. Therefore, the total Del factor required should be equal to the sum of the Del factor for heating, holding and cooling as
The Design Procedure
The design procedure for the estimation of the holding time is as follows:
1. Calculate the total sterilization criterion, Ñ total.
2. Measure the temperature versus time profile during the heating, holding, and cooling cycles of sterilization.
a. For batch heating by direct steam sparging into the medium, the hyperbolic form is used:
3. Plot the values of kd as a function of time.
Example
Solution
During the cooling process, the change of temperature can be approximated as

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FE 462 BIOCHEMICAL ENGINEERING

Cell Kinetics and Fermenter Design
INTRODUCTION
§Understanding the growth kinetics of microbial, animal, or plant cells is important for the design and operation of fermentation systems employing them. Cell kinetics deals with the rate of cell growth and how it is affected by various chemical and physical conditions.
Unlike enzyme kinetics, cell kinetics is the result of numerous complicated networks of biochemical and chemical reactions and transport phenomena, which involves multiple phases and multicomponent systems.
The heterogeneous mixture of young and old cells is continuously changing and adapting itself in the media environment which is also continuously changing in physical and chemical conditions. As a result, accurate mathematical modeling of growth kinetics is impossible to achieve. Even with such a realistic model, this approach is usually useless because the model may contain many parameters which are impossible to determine
GROWTH CYCLE FOR BATCH CULTIVATION

1. Lag phase: The lag phase (or initial stationary, or latent) is an initial period of cultivation during which the change of cell number is zero or negligible. Even though the cell number does not increase, the cells may grow in size during this period.

The length of this lag period depends on many factors such as the type and age of the microorganisms, the size of the inoculum, and culture conditions.

2. Exponential phase: In unicellular organisms, the progressive doubling of cell number results in a continually increasing rate of growth in the population. A bacterial culture undergoing balanced growth mimics a first-order autocatalytic chemical reaction.

The rate of the cell population increase at any particular time is proportional to the number density of bacteria present at that time:

Factors affecting the specific growth rate

Substrate Concentration: One of the most widely employed expressions for the effect of substrate concentration on μ is the Monod equation, which is an empirical expression based on the form of equation normally associated with enzyme kinetics :

The value of Ks is equal to the concentration of nutrient when the specific growth rate is half of its maximum value μmax. According to the Monod equation, further increase in the nutrient concentration after μ reaches μmax does not affect the specific growth rate.

Factors affecting the specific growth rate

Product Concentration: As cells grow they produce metabolic byproducts which can accumulate in the medium. The growth of microorganisms is usually inhibited by these products, whose effect can be added to the Monod equation as follows:

3. Stationary phase: The growth of microbial populations is normally limited either by the exhaustion of available nutrients or by the accumulation of toxic products of metabolism. As a consequence, the rate of growth declines and growth eventually stops. At this point a culture is said to be in the stationary phase.

4. Death phase: The stationary phase is usually followed by a death phase in which the organisms in the population die. Death occurs either because of the depletion of the cellular reserves of energy, or the accumulation of toxic products. Like growth, death is an exponential function. In some cases, the organisms not only die but also disintegrate, a process called lysis.

Modeling of the Bacterial Growth Curve

M. H. ZWIETERING,* I. JONGENBURGER, F. M. ROMBOUTS, AND K. VAN ‘T RIET

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1990, p. 1875-1881

Bioreactors

Stirred Tank Fermenters
A bioreactor is a device within which biochemical transformations are caused by the action of enzymes or living cells. The bioreactor is frequently called a fermenter whether the transformation is carried out by living cells or in vivo cellular components (enzymes).

For a large-scale operation the stirred-tank fermenter (STF) is the most widely used design in industrial fermentation. It can be employed for both aerobic or anaerobic fermentation of a wide range of cells including microbial, animal, and plant cells.

Stirred Tank
Fermenters
Kinetics of Substrate Utilization, Product Formation, and Biomass Production in Cell Cultures
It is difficult to obtain useful kinetic information on microbial populations from reactors that have spatially no uniform conditions. Hence it is desirable to study kinetics in reactors that are well mixed.

Ideal Batch Reactor

Many biochemical processes involve batch growth of cell populations. After seeding a liquid medium with an inoculum of living cells, nothing (except possibly some gas) is added to the culture or removed from it as growth proceeds. Typically in such reactor, the concentrations of nutrients cells, and products vary with time as growth proceeds.
A material balance on moles of component i;

The Ideal Continuous Flow Stirred Tank Reactor (CSTR)
The diagram of this process is shown in fig.2, which is a schematic diagram of completely mixed stirred tank reactor. Such configurations for cultivation of cells are frequently called chemostats.

The Ideal Continuous Flow Stirred Tank Reactor (CSTR)

Productivity of CSTF
MULTIPLE FERMENTERS CONNECTED IN SERIES
A question arises frequently whether it may be more efficient to use multiple fermenters connected in series instead of one large fermenter. Choosing the optimum fermenter system for maximum productivity depends on the shape of the l/rx versus Cx curve and the process requirement, such as the final conversion.

Cell Recycling
For the continuous operation of a PFF or CSTF, cells are discharged with the outlet stream which limits the productivity of fermenters. The productivity can be improved by recycling the cells from the outlet stream to the fermenter.

Alternative Fermenters
Many alternative fermenters have been proposed and tested. These fermenters were designed to improve either the disadvantages of the stirred tank fermenter-high power consumption and shear damage, or to meet a specific requirement of a certain fermentation process, such as better aeration, effective heat removal, cell separation or retention, immobilization of cells, the reduction of equipment and operating costs for inexpensive bulk products, and unusually large designs.

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FE 462 BIOCHEMICAL ENGINEERING
Metabolic Stoichiometry and Energetics
INTRODUCTION
A living cell is a complex chemical reactor in which more than 1000 independant enzyme-catalyzed reaction occur.
The total of all chemical reaction activities which occur in the cell is called metabolism.
Two different type of energy, ligth and chemical, are trapped by inhabitants of the microbial world.The energy obtained from the environment is typically stored and shuttled in convenient high-energy intermadiates such as ATP. The cell uses this energy to perform three types of work: chemical synthesis of large or complex molecules (growth), transport of ionic and neutral substances into or out of the cell or its internal organelles, and mechanical work required fro cell division and motion.
All these processes are, by themselves, nonspontaneous and result in an increase of free energy of the cell.
Metabolic pathways in e.coli
Synthesis of macromolecules from simple nutrients

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FE 462 BIOCHEMICAL ENGINEERING
Immobilized Enzyme

INTRODUCTION
Since most enzymes are globular protein, they are soluble in water. Therefore, it is very difficult or imlpractical to separate the enzyme for reuse in a batch process. Enzymes can be immobilized on the surface of or inside of an insoluble matrix either by chemical or physical methods.
They can be also immobilized in their soluble forms by retaining them with a semipermeable membrane. A main advantage of immobilized enzyme is that it can be reused since it can be easily separated from the reaction solution and can be easily retained in a continuous-flow reactor. Furthermore, immobilized enzyme may show selectively altered chemical or physical properties and it may simulate the realistic natural environment where the enzyme came from, the cell.
IMMOBILIZATION TECHNIQUES
Chemical Method
Physical Method
EFFECT OF MASS-TRANSFER RESISTANCE
The immobilization of enzymes may introduce a new problem which is absent in free soluble enzymes. It is the mass-transfer resistance due to the large particle size of immobilized enzyme or due to the inclusion of enzymes in polymeric matrix. If we follow the hypothetical path of a substrate from the liquid to the reaction site in an immobilized enzyme, it can be divided into several steps
CELL IMMOBILIZATION

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FE 462 BIOCHEMICAL ENGINEERING

Enzyme Kinetics
INTRODUCTION
Enzymes are biological catalysts that are protein molecules in nature.
They are produced by living cells (animal, plant, and microorganism) and are absolutely essential as catalysts in biochemical reactions.
INTRODUCTION
Enzyme reactions are different from chemical reactions, as follows:
1. An enzyme catalyst is highly specific, and catalyzes only one or a small number of chemical reactions. A great variety of enzymes exist, which can catalyze a very wide range of reactions.
2. The rate of an enzyme-catalyzed reaction is usually much faster than that of the same reaction when directed by non-biological catalysts. Only a small amount of enzyme is required to produce a desired effect.
3. The reaction conditions (temperature, pressure, pH, and so on) for the enzyme reactions are very mild.
4. Enzymes are comparatively
Nomenclature of Enzymes
Commercial Applications of Enzymes
1. Oxidoreductases
2. Transferases
3. Hydrolases
4. Lyases
5. Isomerases
6. Ligases
ENZYME KINETICS
Assume that a substrate (S) is converted to a product (P) with the help of an enzyme (E) in a reactor as
The effect of substrate concentration on the initial reaction rate
From these curves we can conclude the following:
1. The reaction rate is proportional to the substrate concentration (that is, first-order reaction) when the substrate concentration is in the low range.
2. The reaction rate does not depend on the substrate concentration when the substrate concentration is high, since the reaction rate changes gradually from first order to zero order as the substrate concentration is increased.
3. The maximum reaction rate rmax is proportional to the enzyme concentration within the range of the enzyme tested.
ENZYME KINETICS
The mechanism of one substrate-enzyme reaction can be expressed as
Michaelis-Menten Approach
If the slower reaction, determines the overall rate of reaction, the rate of product formation and substrate consumption is proportional to the concentration of the enzyme-substrate complex as:
EVALUATION OF KINETIC PARAMETERS
The Michaelis-Menten equation can be rearranged to be expressed in linear form. This can be achieved in three ways:
INHIBITION OF ENZYME REACTIONS
A modulator (or effector) is a substance which can combine with enzymes to alter their catalytic activities. An inhibitor is a modulator which decreases enzyme activity. It can decrease the rate of reaction either competitively, noncompetitively, or partially competitively
OTHER INFLUENCES ON ENZYME ACTIVITY
The rate of an enzyme reaction is influenced by various chemical and physical conditions. Some of the important factors are the concentration of various components (substrate, product, enzyme, cofactor, and so on), pH, temperature, and shear. The effect of the various concentrations has been discussed earlier.
OTHER INFLUENCES ON ENZYME ACTIVITY
Case Study

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FE 462 BIOCHEMICAL ENGINEERING

Introduction-A Little Microbiology
Applications of Microbiology to Foods
Ancient Egyptians used fermentation to produce beer and convert grape juice to wine. They also practiced the aerobic conversion of the alcohol in wine to the acetic acid of vinegar, and the leavening of bread. The present practices of using, for example, pectinases for enhanced release of fruit juices from tissue and amylases for the enzymatic modification of starches, are examples involving the indirect application of microorganisms to foods and food components.
Applications of Microbiology to Foods
The production of xanthan gum by the plant pathogenic bacterium Xanthomonas campestris for use as a viscosity agent in beverages and semisolid food products is an example of the use of an originally undesirable organism for the production of a desirable food and beverage additive.
Applications of Microbiology to Foods
The use of the mold Aspergillus niger to produce high yields of citric acid as a food and beverage acidulant was established in the 1920s and is a classic example of an initial surface culture process that was eventually converted to a submerged aerated process with the use of mutants.
The Nature of Microorganisms
Microorganisms can be found almost anywhere in the taxonomic organization of life on the planet. Bacteria and archaea are almost always microscopic, while a number of eukaryotes are also microscopic, including most protists, some fungi, as well as some animals and plants. Viruses are generally regarded as not living and therefore are not microbes, although the field of microbiology also encompasses the study of viruses.
The Nature of Microorganisms
All microorganisms are allocated to a specific group with respect to growth temperature.
Obligate psychrophiles are defined as those organisms capable of growth at or near 0°C but not at 20°C. Such organisms usually have a maximum growth temperature of 15–17°C.
Psychrotrophic organisms are capable of growth at or near 0°C but exhibit optimum growth at approximately 25°C and are frequently unable to grow at 30°C.
Mesophiles exhibit growth from 20–45°C with an optimum growth temperature usually in the range of 30–35°C. Thermophiles exhibit growth in the range of 45–65°C.
Hyperthermophiles are organisms from oceanic thermal vents and hot springs that are restricted to growth temperatures from 70–120°C. Hyperthermophiles have not yet been isolated from foods.
Nutritional Requirements
All biological systems, from microorganisms to man, share a set of nutritional requirements, which are:

1. Sources of energy

a. Phototrophs organisms which are capable of employing radiant energy.

b. chemotrophs organisms which obtain the energy for their activities and self- synthesis from chemical reactions that can occur in the dark.

2. Sources of carbon

a. autotrophs organisms which can thrive on an entirely inorganic diet, using CO2 or carbonates as a sole source of carbon.

b. heterotrophs organisms which cannot use CO2 as a sole source of carbon but require, in addition to minerals, one or more organic substances, such as glucose or amino acids, as sources of carbon.

3. Sources of nitrogen:

atmospheric nitrogen, inorganic nitrogen compounds, or other derived nitrogen.

4. Sources of sulfur and phosphorus:

elementary sulfur, inorganic sulfur, or organic sulfur.

5. Sources of metallic elements:

sodium, potassium, calcium, magnesium, manganese, iron, zinc, copper, and cobalt.

6. Sources of vitamins.

Physical Conditions
After determining the proper nutrients for the cultivation of bacteria, it is necessary to determine the physical environment in which the organisms will grow best. Three major physical factors to be taken into consideration are temperature, the gaseous environment, and pH.
Since microbial activity and growth are manifestations of enzymatic action, and since the rates of enzyme reactions increase with increasing temperatures, the rate of microbial growth is temperature dependent. Depending on the temperature range over which they grow, bacteria are called psychrophiles, mesophiles, or thermophiles.
Physical Conditions
The principal gases in the cultivation of bacteria are oxygen and carbon dioxide. There are four types of bacteria, according to their response to oxygen:
1. Aerobic bacteria grow in the presence of free oxygen.
2. Anaerobic bacteria grow in the absence of free oxygen.
3. Facultatively anaerobic bacteria grow in either the absence or the presence of free oxygen.
4. Microaerophilic bacteria grow in the presence of minute quantities of free oxygen.
For most bacteria the optimum pH for growth lies between 6.5 and 7.5. Although a few bacteria can grow at the extremes of the pH range, for most species the minimum and maximum limits fall somewhere between pH 4 and pH9.
Culture Media
The growth of microbial population in artificial environments is called cultivation. A culture that contains only one kind of microorganism is a pure culture. A mixed culture is one that contains more than one kind of microorganism. The necessary steps for cultivating microorganisms are:
1. Preparing a culture medium in which a microorganism can grow best.
2. Sterilizing in order to eliminate all living organisms in the vessel.
3. Inoculating the microorganism in the prepared medium.
Sterilization
After a suitable culture medium is selected for the cultivation of a specific microorganism, it is poured into a culture vessel. If you use test tubes or flasks as your culture vessel, the ends of test tubes or flasks should be covered with a suitable closure to allow for the exchange of gases with the atmosphere, yet to keep foreign organisms out of the media. Various types of closures are used in the modern laboratory including cotton plugs, plastic foam, screw caps, metal caps, and aluminum foil.
Sterilization
The medium is then sterilized to eliminate all living organisms in the vessel,-The most common method of sterilization is by moist heat (steam under pressure) in an autoclave. Generally, the autoclave is operated at approximately 15 psi at 121°C. The time of sterilization depends on the nature of the material, the type of container, and the volume. For example, test tubes of liquid media can be sterilized in 15 to 20 minutes at 121°C.
Inoculation
Inoculation is the seeding of a culture vessel with the microbial material (inoculum). The inoculum is introduced with a metal wire or loop which is rapidly sterilized just before its use by heating it in a flame. Transfers of liquid culture are often made by using a sterilized pipette. The inoculation is usually done in a laminar flow hood to minimize the risk of contamination. It is important to know proper pipetting techniques for inoculating or sampling during cultivation.
CELL GROWTH MEASUREMENT
In any biological system, growth can be defined as the orderly increase of all chemical components. To follow the course of growth, it is necessary to make quantitative measurements. Cell growth can be determined by measuring cell number, cell mass, or cell activity.
Measurement of Cell Number
Microscopic Counts: The number of cells in a population can be measured under a microscope by counting cells placed in special counting chambers.
CELL GROWTH MEASUREMENT
Viable Plate Count: A viable cell is defined as one that is able to divide and form a colony.
CELL GROWTH MEASUREMENT
Measurement of Cell Mass
Cell Dry Weight: Cell dry weight can be measured directly by taking an aliquot of cell suspension and centrifuging it. After the supernatant is discarded, the cells are thoroughly washed with distilled water to eliminate all soluble matter. The suspension is recentrifuged and the settled cells are dried in an oven and weighed.
Turbidity: The cell mass can be measured optically by determining the amount of light scattered by a suspension of cells. The technique is based on the fact that small particles scatter light proportionally, within certain limits, to their concentration.
CELL IMMOBILIZATION
As in the case of enzymes, whole cells can be immobilized for several advantages over traditional cultivation techniques. By immobilizing the cells, process design can be simplified since cells attached to large particles or on surfaces are easily separated from product stream. This ensures continuous fermenter operation without the danger of cell washout. Immobilization can also provide conditions conducive to cell differentiation and cell-to-cell communication, thereby encouraging production of high yields of secondary metabolites. Immobilization can protect cells and thereby decrease problems related to shear forces.

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FE 462 BIOCHEMICAL ENGINEERING

Introduction
Some definitions….
Biotechnology: use or development of methods of direct genetic manipulation for a socially desirable goal. Sometimes a broader definition is used, where biotechnology is applied biology.

Biomedical Engineering: engineering on systems to improve human health

Bioengineering, biological engineering: work on medical or agricultural systems, draws on electrical, mechanical, industrial and chemical engineers.

Biochemical Engineering: extension of chemical engineering principles to systems using a biocatalyst to bring about desired chemical transformation.

GENERALIZED VIEW OF BIOPROCESS TYPICAL BIOPROCESS FLOW SHEET
Applications of Biotechnology
The basic questions which need to be asked for the process development and design are as follows:

What change can be expected to occur?
How fast will the process take place?
How can the system be operated and controlled for the maximum yield?
How can the products be separated with maximum purity and minimum costs?
DEFINITION OF FERMENTATION
Traditionally, fermentation was defined as the process for the production of alcohol or lactic acid from glucose (C6H120 6).

A broader definition of fermentation is “an enzymatically controlled transformation of an organic compound” according to Webster’s New College Dictionary (A Merriam-Webster, 1977) that we adopt in this text.

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