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In this term paper we will discuss about biochemical technology.
Micro-organisms have been used in the production of many useful products from time immemorial without even knowing the actual mechanism and principals involved in it. In the wake of modern knowledge, cells and their components are being used in the production of many commercial products for the benefit of human beings at cheaper cost on large scale.
Since all this depends upon living cells activities, a very controlled and specific environment is required which includes highly specific substrate at specific pH and temperature without any impurity for the culture of micro-organisms and the production of required product. The nutrient substrate should be reuseable for cheaper cost of production.
Acid:
An acid is a substance which furnishes H+ (hydrogen) ions by itself or when dissolved in water. This definition is based on classical ionic theory. The strength of an acid solution does not depend upon its concentration but upon the number of free H+ ions present.
According to the new theory of Lowry and Bronsted an acid is a substance which is able to donate proton (H+) or protons. This definition is the most accepted one.
Base:
Based on classical ionic theory a base is a substance which furnishes OH– (hydroxyl) ions when dissolved in water and the basic properties of a base are due to these hydroxyl ions.
Lowry-Bronsted definition of a base is a different one which says – a case is a substance which can accept the proton (H+) or protons.
So, an acid donates proton while a base accepts proton.
Scale of Acidity – pH and pOH Value:
We can express the acidity of a solution on a scale of acidity in the same way as we express the temperature on a thermometer scale.
Concept of pH:
pH or Hydrogen ion concentration is the basis of acid base chemistry which decides the direction of biochemical reactions, because enzymes are active in a particular environment of pH. pH decides the particular structure of protein and thus their reactivity as enzymes.
Acid is a substance which gives H+ (proton) and base accepts this proton. An amphoteric substance can accept and donate protons and thus is neutral.
The neutral pH is 7, below it the solution is acidic and above it, basic, i.e.
According to Bronsted Lowry Concept:
At 25˚ C
Handerson Hasselbatch Equation:
Buffer:
A buffer is an ionic compound which resists changes in its pH value by absorbing a certain quantity of acid or base without undergoing a great variation in pH.
A buffer solution is a mixture of a weak acid HA and its conjugate bases A–, or a mixture of a weak base B and its conjugate acid BH+.
Buffer is used for the calibration of pH meter and to control pH of a medium in which pH dependent chemical activity is taking place, e.g., enzymatic reactions.
Buffer Solutions:
It is known that pure water has a pH value equal to 7. But even the purest form of water cannot retain this value for long. It gradually changes. The same is the case with the solutions of single salts.
In a solution containing a weak acid and the salt of it with a strong base (e.g., CH3COOH + CH3COONa) or a weak base and the salt of it with a strong acid (e.g., NH4OH and nh4ci) possesses few specific properties:
(a) Having a definite pH value
(b) pH value does not alter either on keeping for long or on dilution.
(c) Its pH value is very slightly altered by additions of strong acid in CH3COOH + CH3COONa solution or strong base in NH4OH + NH4CI solution.
Solutions:
Solutions are homogeneous mixtures of two or more substances.
Molar Solution:
It is 1 g molecular weight of a substance dissolved in 1 litre of solution, e.g., 1 M NaCI contains 58.5 g of NaCI in 1 litre of solution.
Normal Solution:
It is 1 g equivalent weight of a substance dissolved in 1 litre of solution, e.g., 1N NaCI contains 58.5 g of NaCI in 1 litre of solution, while 1N H2SO4 contains 49 g of H2SO4 in 1 litre of solution.
(1 g equivalent weight is equal to molecular weight divided by its valence electrons).
Types of Buffers:
Buffers are of various types having a specific range of pH.
For example:
1. Acetate Buffer:
The buffer solution which is prepared by homogeneously mixing acetic acid (Ch3COOH) and sodium acetate (CH3COONa) in varied ratio so as to find a wide range of pH ranging between 3.7-5.6.
2. Borate Buffer:
When boric acid is mixed with borax in varied ratios it makes a buffer ranging in pH from 7.5-9.2.
3. Phosphate Buffer:
When hydrated disodium hydrogen phosphate in mixed with potassium dihydrogen phosphate in varied ratios it makes the solution having pH range between 5.7-8.0.
Buffering Capacity:
The amount of acid or alkali required to produce unit change of pH in the solution is called the buffering capacity of the solution.
For a buffer:
According to Handerson Hasselbalch equation, if the concentration of salt is equal to that of the acid then:
pH = pKa
Then it is called the buffering capacity of the solution. Buffering capacity of a buffer solution can also be defined as the range of the pH between which the solution shows its buffer properties and its buffering effect.
Dimensions:
The physical quantity is indicated with relation to the fundamental quantity or basic quantity.
The dimensions of a physical quantity are the power to which the fundamental units are raised in order to obtain the units of that quantity. Thus dimensions are the fundamental quantities which are used for measurement such as mass, length, time, etc.
For example, velocity is represented by displacement and time:
Putting the fundamental quantities in the same equations:
Thus [LT-1] is the expression of a physical quantity called dimensional formula of velocity.
Dimensional Quantities:
Units:
The physical quantity is expressed in the form of fundamental quantities. Each fundamental quantity is expressed in the form of unit. Thus physical quantities also bear a specific unit.
For example, velocity is expressed as unit of displacement covered per unit time; therefore, its unit is metre per second.
SI Unit:
The measurements of all the systems of weight, length, etc., are linked to an international system called the International System of units or SI units. It began in the month of October 1960.
Physical Variables:
Biochemical process requires calculations at times using measurable physical variables like length, area, etc., but there are some which cannot be measured but felt, like smell etc. Physical quantities may have units called substantial variable or without unit but expressed as ratios, e.g., specific gravity.
Physical variables can be divided into two types:
1. Substantial Variables:
These variables have a unit. These variables are measured against a particular physical standard. These standards are called units. For e.g., mass, length, time, etc.
2. Natural Variables:
The variables which are dimensionless are grouped as natural variables. Thus these variables do not have any unit as well. For e.g., refractive index, specific gravity, etc.
Concept of Probability:
Probability:
Probability is derived from the word “to probe” or “to find out”. It was applied to those, which are not easily accessible or understandable. The origin of probability was in 6tri century when it was applied to games of chance and gambling.
Blaise Pascal and Pierre de Ferment added it as the branch of Mathematics in the 17th century. These days various theories of probability and probabilistic modeling are used in almost all the fields like business, industry and sciences such as telephone exchange, computer process, genetics, etc. It tells about the chances of occurrence of a particular phenomenon.
It is classified as:
(i) Theoretical Probability:
It is based on mathematical calculations of the occurrence of an event or phenomenon under ideal conditions.
(ii) Experimental Probability:
The actual results obtained by repeated testing and observation.
Sampling:
It is collection of elements for the component of the sample. It may be done randomly or systematically, for example, constant skipping based on taking every nth reading from the random population or stratified for example, sample collection from different strata of population.
Sampling can be achieved by two major techniques:
1. Quadrat Sampling:
This is the technique in which sampling is done by using quadrant. Sometimes a few problems occur such as – one may not be able to count how many individual plants there are in a dense dump.
To solve this problem following methods are used:
(a) Estimating plant density by counting the number of individuals per unit area.
(b) Estimating plant frequency by looking at the distribution of individuals per unit area.
(c) Assessing plant cover by measuring the proportion of the ground covered by a plant species.
2. Transect Recording:
A transect is an imaginary line along which one makes careful and systematic observations.
Transect can be of two types:
(a) Belt Transect.
(b) Line Transect.
Transect recording can be used to study how communities and ecosystems change along an environmental gradient, e.g., through a woodland, along a rocky shore or up the side of a hill.
Fluid Flow and Fluid Mixing:
Fluid Flow:
Fluid dynamics is a part of fluid mechanics and deals with fluid flow i.e., motion of liquids and gases in natural conditions. It has several branches including aerodynamics (or the study of air and other gases in motion) and hydrodynamics (or the study of liquids in motion).
Fluid dynamics offers a systematic structure derived from flow measurement and used to solve practical problems. The solution to a fluid dynamics problem typically involves calculating various properties of the fluid such as velocity, pressure, density and temperature as functions of space and time.
When the fluid flows without any hindrance then flow is considered to be a steady flow. Steady- state flow refers to the condition where the fluid properties at a point in the system do not change over time. Otherwise, flow is called unsteady or transient flow. For example, laminar flow over a sphere is steady with respect to the sphere which is stationary. Turbulent flows are unsteady by definition.
Steady flows are easily observed and studied as compared to unsteady flows.
The fluids may be of any of the two categories depending upon Newton’s law of viscous flow.
During large scale fermentation the viscosity of fluid changes from Newtonian to non-Newtonian type which causes limitation in growth of cells due to limited nutrient and oxygen supply. In aerobic processes, to overcome the less solubility problem of oxygen, special devices like airlift fermentor, bubble column fermentor, etc., are used.
Fluid Mixing:
Fluid mixing involves two mechanisms – diffusion and advection (advection is a transport mechanism of a substance or conserved property by a fluid due to the fluid’s bulk motion). In liquids, molecular diffusion alone is hardly efficient for mixing. Advection is the transport of matter by a flow and is required for better mixing. When considered at micro level fluid mixing behaves radically different.
The range of sizes varies from 2 millimetres to the nanometre level. At this size range normal convection does not happen unless you force it. Diffusion is the dominate mechanism where two different fluids come together through their movement from their higher concentration level to their lower concentration level. Diffusion is a relatively slow process.
Mass Transfer:
Mass transfer is the net movement of mass from one location usually meaning a stream, phase, fraction or component to another. Mass transfer occurs in many processes, such as absorption, evaporation, adsorption, drying, precipitation, membrane filtration and distillation. Mass transfer is used by different scientific disciplines for different processes and mechanisms. The phrase is commonly used in engineering for physical processes that involve diffusive and convective transport of chemical species within physical systems.
It refers to the transfer of oxygen from outside into the cell through fermentation medium during a bioprocess in a bioreactor. The main hurdle in this transfer is the gas liquid interface which decreases the uptake of oxygen by the cells.
The problem has been solved by:
(i) Agitating the medium which increases the surface area by forming small air bubbles and decreasing the thickness of the liquid film at the gas liquid interface.
(ii) Using mixing devices like impeller, sparger and baffles which by proper mixing provide homogenous environment so that oxygen and nutrients become available to the cells.
Heat Transfer:
Sufficient amount of heat is generated during various steps in fermentation which is required to be controlled for efficient performance of the biological process. Temperature is measured by using mercury glass thermometer, bimetallic thermometer, pressure bulb thermometer, thermocouple thermometer or metal resistance thermometer.
On the basis of heat evolution rate, desired temperature of heat transfer area required can be calculated. The bioreactors are thus equipped with heating/ cooling devices for keeping the optimum temperature. This is achieved by circulating steam or chilled water in jackets fitted around the fermentor. These devices are also used for the sterilization of the fermentor in the beginning of the process and also for cleaning the vessels of fermentor.
Homogenous Reactions:
They occur in an optimum environment during a biochemical process. For these reactions various factors should be carefully maintained because growth kinetics is greatly influenced by these factors.
(a) Growth Media:
A growth medium is selected or formulated considering the following points:
(i) Should provide required nutrients in balanced form.
(ii) Maximum yield.
(iii) Minimum wastage.
(iv) Low cost.
A medium is designed, considering growth dependent products which may be:
(i) Primary metabolites produced during exponential phase of growth.
(ii) Secondary metabolites are produced after log phase/exponential phase of growth.
A medium should be cheap and provide all the required nutrients to cells. For this purpose wastes like sugarcane bagasse, whey, etc., which can provide both energy carbon and other minerals required for growth and produce least pollution are suitable.
(b) Growth Kinetics and Fermentation process:
Growth and metabolism of microbes is also important for the production of product in a bioprocess. The genetic engineering is thus important for the development of improved strains of organisms (microbes). Growth kinetics and fermentation process determines the culture mode.
Four fundamental culture modes are used in industrial process like fermentation, these are:
(i) Batch culture,
(ii) Fed batch culture,
(iii) Semi continuous culture, and
(iv) Continuous culture.
Batch Culture:
It has limited amount of nutrient and there is no addition or removal of nutrient during the process.
The growth curve obtained is sigmoid which has five phases:
(i) Lag Phase:
Lag phase or time of adaptation or no growth, it is non-productive and thus should be of minimum duration.
(ii) Log Phase:
Log phase or trophophase/exponential growth is the stage when the microbes grow faster.
(iii) Deceleration Time:
Decrease in growth rate because of reduction in nutrient and production of excretory waste (antibiotics).
(iv) Stationary Phase:
Cell growth stops as no more nutrients is present in the medium.
(v) Death Phase:
Cells start to die.
Effect of temperature on Growth:
Temperature affects enzymatic activities of the organisms and thus their growth due to influence on conversion of carbon to cell-mass.
Accordingly organisms have been classified as:
(i) Psychrophile:
Optimums temperature 15 ± 2°C
(ii) Mesophiles:
Optimum temperature 35 + 5°C
(iii) Thermophile:
Optimum temperature 60 ± 5°C
(iv) Hyper thermophile:
Optimum temperature 93 ± 5°C
Effect of substrate concentration on growth:
Low concentration of a substrate limits growth and is given by the equation.
µ = Specific growth rate
S = Residual substrate concentration
Ks = Substrate Constant
µmax = maximum possible growth rate.
On increasing the substrate concentration µ increases initially but further increase becomes inhibitory. Thus growth is inversely proportional to concentration of the substrate.
(c) Growth associated and non-growth associated products:
In growth associated bio process, product concentration directly depends on cell concentration. Products are formed from primary energy metabolism of the organisms thus with the increase in cell growth, product formation also increases.
In non-growth associated process, the products are produced when cell growth retards and are called secondary metabolites. The accumulation of these products causes cell death.
Stoichiometry:
During biochemical reactions new groups of atoms and molecules are formed through the rearrangement of atoms and molecules. By stoichiometry, the relationship of mass and moles of reactant and products are measured, provided equation and atomic weights of reactant are correctly written. If we consider the cell growth in a culture medium, then some reactants are provided more than the requirement, e.g., carbon which is required for biosynthesis and energy generation both.
Carbon requirement under aerobic condition may be estimated from cellular coefficient (Y).
Y = Quantity of cell dry mass produced/Quantity of carbon substrate used