14863 chapter 23 biotechnology

Engineering Chemistry Copyright 2013 Wiley India Pvt. Ltd. All rights reserved.

Learning Objectives

1.Biotechnology, its definition and significance.

2.Applications of biotechnology – in agriculture, food science, medicines, as transgenic plants and animals and in environment.

3.Bioreactors and its types.

4.Fermentation process and production of alcohol and vitamins.

5.Properties of enzymes and their applications in the industries.

6.Basic instrumentation, types and applications of biosensors.

7.Types of biofertilizers and their advantages and disadvantages.

8.Biosurfactants and intermolecular multiple force theory (IMMFT)

9.Biochips and its applications.


Introduction

Biotechnology is defined as the use of scientific and engineering principles to manipulate organisms or their genes, producing one or more of the following:

1.Organisms with specific biochemical, morphological, and/or growth characteristics.

2.Organisms that produce useful products.

3.Information about an organism or tissue that would otherwise not be known.

The emergence of biotechnology as rapidly developing area of research with tremendous applications in healthcare, environment, food industries, etc., has amplified its significance manifold in day-to-day lives of the people. One of the major reasons why biotechnology has gained significance is because of gene cloning. Although most of the useful products can be obtained from microorganisms, the list is limited to those synthesized naturally.


1. Some important pharmaceutical products can be produced by higher organisms but not by microbes. This has been changed by the application of gene cloning to biotechnology.

2. The gene for an important animal or plant protein is taken from its host and introduced into a bacterium (Figure 1) leading to the production of recombinant protein. This can later on be obtained in large amounts.


Figure 1 General scheme of production of animal protein by a bacterium. (mRNA stands for messenger

ribonucleic acid).

Applications of Biotechnology

Recent developments have led to clear understanding of biotechnology and has helped us to apply biotechnology in improving the quality of life. The following are the important applications of biotechnology.

Agriculture

1.Increases in agricultural production can come mainly from the development of high-yield strains of crops. During the twentieth century, rapid strides were made in increasing the production of crops per unit area.

2.The green revolution is the name attached to post–World War II programs that have led to the development of new strains of crops with higher yields, better resistance to disease, or better ability to grow under poor conditions.

3.However, these improved crop varieties required greater use of fertilizers and pesticides and water. In some cases, the crop was not considered desirable to eat.


This has led to the genetic modification of crops, with major implications for agriculture. The development and use of genetically modified organisms (GMOs) has given the promise of increased agricultural production. There is considerable interest in the potential for genetic engineering to develop strains of crops with entirely new characteristics such as:

1.To develop new crops with the same symbiotic relationship found in legumes (members of the pea family) so that they “fix” nitrogen (convert atmospheric gaseous nitrogen to a form that can be used by green plants). This in turn will reduce the cost of fertilizers.

2.To develop strains with improved tolerance of drought, cold, heat, salts and toxic metals.

3.To make the plants herbicide resistant.

4.To develop crops with improved nutritional value.

5.To reduce the use of pesticides by developing pest resistant plants.

6.To develop virus resistant plants.


Development of of crops with increased nutritional value

Rice with beta carotene- gene taken from daffodils (modification requires the introduction of four secific genes)

Development of pest resistant plants

The bacterium Bacillus thrugienesis (Bt.) – cry gene- produced protein can kill pests likeworms beetles and mosquitoes

Food Science

1.The use of microorganisms in making bread, cheese, and wine is as old as civilization itself. In bread, yeast is used as a leavening agent – that is, to produce gas that makes the dough rise.

2.A particular strain of Saccharomyces cerevisiae is added to a mixture of flour, water, salt, sugar, etc.

3.A genetically engineered strain of “fast-acting” yeast cuts the rising time of most breads to half. Besides, the application of biotechnology extends to production of alcoholic beverages.

4.The protein rennin is used in making cheeses. Prior to the advent of genetic engineering, rennin was extracted from the fourth stomach of cattle.

5.Genetically engineered bacteria are now used for the commercial production of rennin.


Medicines

The advancement of biotechnology has seen tremendous application in the field of healthcare leading to improved and early diagnosis and production of engineered proteins.

1.Molecular Diagnosis- PCR (polymerase chain reaction)

2.Gene Therapy

3.Production of Genetically Engineered Proteins


Transgenic Animals and Plants

The uses of transgenic animals are as follows:

1.To study of gene expression in mammals.

2.To serve as an excellent model system for study of diseases.

3.To enhance growth hormone levels that might result in leaner pigs with improved meat quality and with faster growth. Similar experiments were done with chicken and fish.

4.To produce virus resistant strains of chicken that will have higher commercial value.

5.To produce and secrete biological products such as valuable proteins in milk.

6.To study safety of vaccines before using them on humans.

7.To test the safety of chemicals by producing transgenic animals that are more sensitive to these chemicals than the non-transgenic animals.


The transgenic plants obtained through genetic engineering have several benefits. Some of these are listed below:

1.To serve as model organisms to study molecular biology.

2.To improve product quality.

3.To improve resistance to biotic and abiotic stresses.

4.To produce novel products such as insulin, antibodies, etc.

5.To produce a protein from a gene coding a protein from an antigen that can act as an edible vaccine.


Environment

1.The solution that biotechnology can offer in control of environmental pollution is degrading the biodegradable pollutants with the help of microorganisms.

2.In waste water treatment, biofilters are used that consist of microorganisms. These microorganisms then convert the harmful pollutants in the gases to less harmful or harmless substances.

3.The microorganisms carry out aerobic or anaerobic fermentation of the pollutants present in the liquid wastes. The solid wastes are subjected to anaerobic digestion by the microorganisms.

4.Petroleum and oil spills in oceans can be degraded by genetically engineered bacteria.

5.The use of biotechnology in environmental pollution control extends to isolation and development of strains that have improved capabilities of biodegradation of harmful compounds.


Fermentation

The metabolism of glucose or another sugar by glycolysis is a process carried out by nearly all cells. One process by which pyruvic acid is subsequently metabolized in the absence of oxygen is fermentation. Fermentation is the result of the need –to recycle the limited amount of nicotinamide adenine dinucleotide (NAD) by passing the electrons of reduced NAD off to other molecules.

There are many kinds of fermentation pathways (Figure 2).


Figure 2 Fermentation Pathways

Production of Alcohol

In alcoholic fermentation (Figure 3), carbon dioxide is released from pyruvic acid in presence of enzyme pyruvate decarboxylase to form the intermediate acetaldehyde, which is quickly reduced to ethyl alcohol by electrons from reduced NAD. Alcoholic fermentation, although rare in bacteria, is common in yeasts and is used in making alcoholic beverages.

Engineering Chemistry Copyright 2013 Wiley India Pvt. Ltd. All rights reserved.Figure 3 Alcoholic Fermentation

Production of Vitamins

Vitamins are organic molecules that the body needs in very small, but necessary, amounts. Many vitamins are components of the enzyme systems that catalyze metabolic reactions. They are known as micronutrients.

Table 1 Some important vitamins, their uses and microorganisms producing them

The ability of microorganisms to make vitamin B12 and

riboflavin is an example of highly successful amplification of microbial synthesis. Engineering Chemistry

Copyright 2013 Wiley India Pvt. Ltd. All rights reserved.

Bioreactors

A bioreactor is a vessel in which the living cells or enzymes utilize the substrate (generally of low value) to form products that are of high commercial value. An important task in biotechnological industry is to manipulate regulatory mechanisms so that the organisms continue producing large quantities of useful substances for humans. Industrial microbiologists accomplish this in several ways:

1.by altering nutrients available to the microbes,

2.by altering environmental conditions,

3.by isolating mutant microbes that produce excesses of a substance because of a defective regulatory mechanism, and

4.by using genetic engineering to program organisms to display particular synthetic capabilities.


The following provisions are made in a bioreactor to facilitate the process:

1.Aeration: This is required when aerobic fermentation needs to carried out.

2.Agitation: This is needed for mixing the cells with the medium or substrate. Generally, stirrers are provided to facilitate agitation. The medium is usually synthetic and chemically defined.

3.Regulation: This is required to control the parameters such as pressure, temperature, pH, aeration, etc.

4.Sterilization: This is required to prevent contamination.


Types of Bioreactors

The main types of bioreactors are:

1.Stirred tank reactor,

2.Bubble column reaction,

3.Air lift reactor,

4.Fluidized bed reactor,

5.Packed bed bioreactor and

6.Photo bioreactors.


Depending on the process performed, the bioreactors can be classified as follows:

1.Aerobic fermenters: In this, sterile air is required to carry out the process as it is carried out by aerobic microorganisms.

2.Anaerobic fermenters: In this, aeration is generally not required and the process is carried out by anaerobic microorganisms.

3.Solid state fermenters: In this, the solid substrates are used to carry out the fermentation as in mushroom cultivation.

4.Immobilized cell bioreactors: In this, the cells are immobilized on cellulose, dextran, glutaraldehyde, polymer matrices, etc., depending on the requirement. Cell immobilization is beneficial when the product formed is of very low molecular weight or in case of intracellular enzymes.Engineering Chemistry


Previously, the microorganisms were grown in large vessels from which the product was purified after the cells were removed [Figure 4(a)].

This was known as batch culture method. It has now been replaced by continuous culture method that makes use of a fermenter or bioreactor in which fresh medium is introduced on one side and medium containing the product is withdrawn on the other side [Figure 4(b)].

Continuous reactors, which operate under strictly controlled temperature and pH conditions, are used in many types of industrial fermentations.


Figure 4 Two different systems for the growth of microorganisms: (a)

batch culture, (b) continuous culture.

Enzymes and their Applications in Industry

Properties of Enzymes

Enzymes are a special category of proteins found in all living organisms. The following are the important properties of enzymes:

1.They act as catalysts—substances that remain unchanged while they speed up reactions to as much as a million times the uncatalyzed rate, which is ordinarily not sufficient to sustain life.

•The reaction rate can be increased by increasing the temperature.

•However, most cells would die when exposed to such a rise in temperature.

•Thus, enzymes are necessary for life at temperatures that cells can withstand.


2. Enzymes lower the activation energy (the energy to start the reaction) so reactions can occur at mild temperatures in living cells (Figure 5).

Figure 5 The effect of enzymes on activation energy.

3. Enzymes also provide a surface on which reactions take place.

4. Enzymes generally have a high degree of specificity; they catalyze only one type of reaction, and most act on only one particular substrate.


Industrial Applications of Enzymes

All enzymes used in industrial processes are synthesized by living organisms. With a few exceptions, such as the extraction of the meat tenderizer papain from papaya fruit, industrial enzymes are made by microorganisms. Enzymes are especially useful in industrial processes because of their specificity. They act on a certain substrate and yield a certain product, thus minimizing problems of product purification.

The following are the important industrial uses of enzymes:

1.Proteases, which degrade proteins and are added to detergents to increase cleaning power, are made industrially by molds of the genus Aspergillus and bacteria of the genus Bacillus. Proteolytic enzymes have also been added to drain cleaners, where they are especially useful in degrading hair, which often clogs bathroom drains.

2.Amylases, which degrade starches into sugars, also are made by Aspergillus species. Engineering Chemistry


3. Lipases from the yeast Saccharomycopsis is also important as a degradative enzyme that degrades fats. A drain cleaner with a lipase should do a good job on kitchen drains.

4. Invertase (glucose isomerase) from Saccharomyces converts glucose to fructose, which is used as a sweetener in many processed foods.

5. To make high-quality paper, much of the lignin, a coarse material in wood, is removed by expensive chemical means to produce a purer cellulose wood pulp. The fungus Phanerochaete chrysosporium secretes enzymes that digest both lignin and cellulose. If the enzymes can be separated and purified, one might be used selectively to digest lignin, leaving cellulose unchanged. Another lignin-digesting enzyme has been identified in a strain of Streptomyces bacteria.


Biosensors

A device using the interaction of a biological material (e.g., enzymes, antibodies, nucleic acids, whole cells, tissues or organs) with an analyte (a compound whose concentration needs to be determined) to measure the detectable physical change is known as a biosensor. The detectable change is converted into an electric signal by a transducer and is further amplified and displayed.

The interaction between the analyte and the biological material can be of two types:

1.Affinity biosensors: Binding of the analyte to the biological material.

2.Catalytic biosensors: Conversion of analyte leading to the formation of a new molecule.


Basic Biosensor Instrumentation

A biosensor is made up of two parts:

1.Physical component: Transducer, amplifier, display unit, etc.

2.Biological component: Enzymes, antibodies, nucleic acids, etc.

Figure 6 Block diagram of a biosensor. Engineering Chemistry Copyright 2013 Wiley India Pvt. Ltd. All rights reserved.

Types of Biosensors

Based on the type of physical change produced, the biosensors are of following types:

1.Potentiometric biosensors: Measure the electrical potential.

2.Calorimetric biosensors: Measure the heat released or absorbed by a reaction.

3.Optical biosensors: Measure the light produced or absorbed during the reaction.

4.Acoustic wave biosensors: Measure the change in the mass of the biological material during the reaction.

5.Amperometric biosensors: Measure the movement of electron during an oxidation-reduction reaction.


Applications of Biosensors

Biosensors find a widespread application in the following:

1.Medical diagnosis and healthcare.

2.Quality control in pharmaceutical industries, agriculture and food industries.

3.Quality control of the environment by monitoring pollutants and industrial effluents.

4.Biological warfare agents.


Biofertilizers

Biofertilizers are defined as bacteria, algae and fungi (single or in combination) on biologically active products that help in enriching the soil nutrients.

Table 2 Some important microorganisms used as biofertilizers


Advantages of Biofertilizers

1.Inexpensive to produce.

2.Relatively simple production technology.

3.Provide soil enrichment.

4.Eco-friendly.

5.Sustainable for long-term use.

Disadvantages of Biofertilizers

1.The amount of nutrients provided is not sufficient for all crops.

2.The process is slow and a lot of time is required to produce significant results.


Biosurfactants

The compounds that decrease the surface tension of a liquid, the tension at the interface between two liquids, or between a liquid and a solid, are known as surfactants. These are usually amphiphilic organic compounds containing hydrophobic tails and hydrophilic heads [Figure 7(a)]. A surfactant contains both, an oil-soluble and a water-soluble component. In the case where water is mixed with oil, surfactants adsorb at the interface between water and oil [Figure 7(b)]. Detergents, wetting agents, emulsifiers, foaming agents, dispersants are all examples of surfactants.


Figure 7 (a) Surfactant showing hydrophilic

head and hydrophobic tail and (b) oil droplet

stabilized by surfactant.

Classification of Biosurfactants

Biosurfactants are classified into five broad groups based on their chemical composition. These are:

1.Glycolipids

2.Lipopeptides and lipoproteins

3.Phospholipids

4.Hydroxylated and cross-linked fatty acids

5.Polymeric and particulate surfactants


Advantages of Biosurfactants

1.Biosurfactants augment the emulsification of hydrocarbons, while having the potential to solubilize hydrocarbon contaminants and enhancing their availability for microbial degradation.

2.Biological treatments terminate pollutants effectively, while being biodegradable themselves. Hence, biosurfactant producing microorganisms are used extensively in the accelerated bioremediation of hydrocarbon-contaminated sites. They are therefore environment friendly.

3.It is possible to produce them on a large scale.

4.They are diverse and selective in nature.

5.They can carry out their activities under severe temperatures and pH values.


Applications of Biosurfactants

1.Enhanced oil recovery.

2.Formulation of herbicides and pesticides.

3.Formulation of detergents.

4.In medicine and healthcare because of their antimicrobial properties.

5.In paper and pulp and textile industries.

6.In food industries.


Intramolecular Multiple Force Theory (IMMFT) of Biosurfactants

1.The supramolecular structures are self-assembled structures consisting of two or more molecules, stabilized, guided and governed by intermolecular interactions rather than by traditional covalent bonds.

2.Intramolecular multiple force theory (IMMFT) deals with the forces that develop in different domains of similar supramolecules.

3.The supramolecule, such as dendrimer, has many discrete zones that have their own environment with center of forces.

4.When these forces are coordinated with each other in similar molecules, intramolecular multiple force develops.


Biochips and its Applications

A biochip is an assembly of miniaturized test sites (microarrays) arranged on a solid substrate that facilitates many tests to be performed simultaneously in order to accomplish higher through put and speed.

It is ananalytical device that contains sensors and biomolecules that have been immobilized either on the planar surface or in microarrays.

The construction of a biochip involves in-depth knowledge of nanotechnology. The potential of biochips can be tapped in the following fields:

1.Genomics: The use of biochips in genomics facilitates automated genomic analysis, genotyping, DNA amplification, DNA hybridization assays, while improving speed and accuracy.

2.Proteomics: Biochips are used in protein investigative studies for multi-dimensional micro-separations, electrokinetic sample injections and pre-concentration methods such as stacking.


3. Cellomics: The use of biochips assists in the study of drug-cell interactions for drug discovery and biosensing. Biochips are especially useful in the handling of single of few cells with nanoprobes in carefully controlled environments.

4. Biodiagnostics: Biochips are used for genetic diagnostics and development of biosensors involving optimization of the platform, decrease in detection time and enhancing signal-to-noise ratio. Protein biochips have their surfaces modified to detect various disease causing proteins simultaneously. Protein and receptor specific antibodies are fixed to the biochips to separate and analyze the proteins, in order to help find a cure for diseases. Similarly, DNA and carbohydrate chips can also be used.


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14863 chapter 23 biotechnology