Chapter 1: Historical Background of Biotechnology
In the mid-1850s, in the French industrial town of Lille, a distillery owner was losing money he could not afford to lose. His factory fermented sugar-beet juice into alcohol, and for years the process had worked. Now, batch after batch was going wrong. Instead of alcohol, the vats were turning sour. Nobody could say why. Two vats treated in exactly the same way, on the same day, with the same juice, would give two completely different results. The owner approached a young chemistry professor at the local university — Louis Pasteur — and asked him to find out what was going wrong. Pasteur did something that chemists of his time did not usually do with an industrial vat: he took drops from the good vats and the spoiled vats and put them under a microscope. In the healthy vats he saw plump, rounded yeast cells. In the sour vats those cells were fewer, and among them were tiny rod-shaped bodies that had no business being there. Fermentation, he concluded, was not a purely chemical breakdown happening on its own. It was the work of living organisms — and if the wrong organism got in, you got the wrong product. That single insight, born on a factory floor rather than in a laboratory, is where modern biotechnology begins.
Diagram
Fig. 1.1 – Split illustration for the chapter opening: on the left, a 19th-century distillery vat with a simple microscope beside it showing round yeast cells and rod-shaped bacteria; on the right, a modern stainless-steel fermenter with a control panel, joined by an arrow labelled “from craft to controlled process”
This chapter will answer each of these questions — and by the end of it, the vats in Lille and the pot of milk in your kitchen will look like two versions of the same idea.
1.1 What is Biotechnology?
The word biotechnology is built out of two smaller words — “bio”, from biology, the study of living things, and “technology”, the practical use of knowledge to make something useful. Put together, biotechnology is the study and use of living systems to make goods and provide services that people need.
That combination is the key to the whole subject. A living organism is not simply being observed here, as it would be in a biology class. It is being put to work. The yeast in a fermenter, the bacterium in a vat of milk, the mould that yields an antibiotic — each is a tiny chemical factory, and biotechnology is the discipline that learns to run those factories deliberately.
A New Name for an Old Practice
Biotechnology is often described as a new discipline built on very old practices, and that description is exact. Long before anyone knew that microorganisms existed, people across the world were making curd, cheese, vinegar, wine, bread and beer. Records suggest wine and beer were being made as early as about 6000 BC, and bread and curd by about 4000 BC.
Those early producers had recipes, not explanations. They knew that a little old curd added to warm milk would set fresh curd by morning, but they did not know that a living organism was carrying out the change. The practice was correct; the reason was missing. Biotechnology as a named subject began only when the reason was finally supplied and could be controlled.
1.1.1 Karl Ereky and the Coining of the Term
The term Biotechnology was given by Karl Ereky (Károly Ereky), a Hungarian agricultural engineer, in 1917. It appeared in print in 1919 in his book Biotechnologie der Fleisch-, Fett- und Milcherzeugung im landwirtschaftlichen Grossbetriebe — Biotechnology of Meat, Fat and Milk Production in the Large-Scale Agricultural Farm.
Ereky was not writing about laboratories. He was writing about farming on an industrial scale, and his central question was a practical one: how efficiently can a cheap raw material — animal feed such as sugar beet — be converted by living animals into valuable products such as meat, fat and milk? Seen that way, a farm is a conversion plant and the animals in it are the converting machinery.
According to Karl Ereky, “biotechnology is a process by which raw material could be biologically upgraded into socially useful products.”
Notice the word upgraded. It carries the whole idea: something of low value goes in, a living system acts on it, and something of higher value comes out. That is as true of a modern fermenter making insulin as it was of Ereky’s pigs. For coining the term and framing this idea, Karl Ereky is regarded by one group of scientists as the Father of Biotechnology.
1.1.2 The EFB and OECD Definitions
As biotechnology grew into an international industry, scientific and economic bodies framed formal definitions of their own. Two of these are important for you, because they are the ones the subject is formally defined by.
The European Federation of Biotechnology (EFB) defines biotechnology as “the integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services.” In plainer words, biotechnology is the integrated use of biochemistry, microbiology and engineering sciences in order to achieve the technological application of the capabilities of microorganisms, cultured tissues, cells and parts thereof.
Two phrases in that definition are worth pausing on. “Parts thereof” means that you need not use a whole organism — an isolated enzyme is enough, which is why a washing powder containing a bacterial enzyme is a biotechnological product. “Molecular analogues” means laboratory-made molecules that behave like natural ones, such as a synthetic DNA sequence.
The Organization for Economic Co-operation and Development (OECD) defines biotechnology as “the application of science and technology to living organisms as well as parts, products and models thereof, to alter living or non-living materials for the production of knowledge, goods and services.”
The OECD wording is the broadest of the three. It allows the material being altered to be non-living — as when bacteria are used to break down an oil spill or clean industrial effluent — and it counts the production of knowledge, not only of goods, as an outcome of biotechnology. A DNA sequencing project that produces no saleable product at all still falls within this definition.
Biotechnology in Its Modern Sense
Taking the three definitions together, biotechnology in its modern sense is the integration of the natural sciences — biochemistry, microbiology and engineering among them — with living organisms, cells, parts of cells, and molecular analogues, in order to apply science and technology to the alteration of living or non-living materials, and so to produce knowledge, goods and services.
That single sentence is worth unpacking as a working picture of what the subject actually does, because every later chapter of this book fits somewhere inside it. The relationship is easiest to see as a flow.
Flowchart
FC 1.1 – Biotechnology as a conversion process: raw material (feedstock, milk, plant biomass, waste) → living system at work (whole organism, cell, tissue, isolated enzyme or molecular analogue) → controlled conditions supplied by engineering (temperature, pH, oxygen, sterility, scale) → useful output (goods, services, knowledge)
No single science can supply all four of those boxes. Biology identifies the organism and explains what it can do; biochemistry explains the reactions it carries out; microbiology keeps the culture pure and healthy; chemical engineering builds the vessel and holds the conditions steady at a scale of thousands of litres. This is why biotechnology is called an interdisciplinary subject, and why a biotechnologist is expected to be comfortable moving between a microscope and a set of process controls.
Diagram
Fig. 1.2 – A wheel diagram with “Biotechnology” at the centre and spokes to the contributing disciplines: biochemistry, microbiology, genetics, cell biology, chemical engineering, immunology and bioinformatics, each spoke labelled with one contribution it makes
1.2 Biotechnology in Ancient Civilisations
Biotechnology is as old as human civilisation itself. Every settled society that has been studied turns out to have used living organisms to change one material into another — to raise bread, to sour milk, to turn fruit juice into wine, to keep a vegetable edible through a winter. None of these societies could see a microorganism. All of them had learnt to use one.
This is why biotechnology is sometimes called a newly named discipline built on age-old practice. The techniques came first, by trial and error, over thousands of years. The explanation came last.
Domestication: The First Deliberate Change
The oldest form of biotechnology is not fermentation but farming. Roughly ten thousand years ago, human communities began to domesticate plants and animals rather than only hunting and gathering them.
Early farmers noticed that not all plants of a kind were alike. Some gave larger grain, some tasted better, some survived a disease that killed their neighbours. By saving seed from the best plants and sowing it the next season, and by repeating this over many years, they gradually built up varieties that carried the traits they wanted — disease resistance, better taste and higher yield. With domesticated animals, the same principle was applied: the animals with the desired traits were bred, generation after generation.
This is selective breeding, and it is a genuine biotechnology even though no laboratory is involved. The farmer is altering the inherited make-up of a population to obtain a more useful product — exactly the “biological upgrading” described in the previous section, carried out with nothing more than patience and observation.
Microorganisms Put to Work
Alongside farming, ancient societies were using microorganisms for two purposes that mattered enormously before refrigeration existed: preservation and improvement of food.
For centuries, yeasts, moulds and Lactobacillus bacteria were used to preserve milk, fruits and vegetables. A fermented food keeps far longer than the fresh material it was made from, and it is often easier to digest and richer in flavour. Wine and beer were made by peoples who understood brewing and fermentation as procedures, and the baking of cakes and bread was practised by the people of the Middle East.
Records place the development of wine and beer as early as about 6000 BC, and the making of bread and curd as early as about 4000 BC.
Diagram
Fig. 1.3 – A simple world map with labelled markers for the early fermentation centres discussed: Mesopotamia (Babylonians and Sumerians — beer, vinegar), Egypt (leavened bread, beer), the Indus Valley and Indo-Aryan region (fermented foods and beverages, medicines), and the Middle East (baking); each marker carrying the product and an approximate period
Fermented Products Across Ancient Civilisations
In India. Ancient Indian texts describe somarasa, a beverage obtained from certain plants and plant materials. The very existence of such a preparation shows that people of the period knew how to obtain an alcoholic drink from more than one kind of plant source — that is, they had a working method of brewing rather than a single lucky recipe.
The Indus Valley and Indo-Aryan civilisations practised biotechnology in three distinct ways: to produce fermented foods, to prepare medicines, and to keep their surroundings clean. That last use is worth noticing, because treating waste by letting microorganisms break it down is precisely what a modern sewage treatment plant does.
In Mesopotamia and Egypt. The Babylonians, Sumerians and Egyptians all practised fermentation, and they used it to prepare alcohol and vinegar. They also obtained a gas from the process — mainly carbon dioxide — which is what gives leavened bread its rise. Yeast was used for producing wine and beer, while bacteria were used to produce yoghurt.
Why the Ancient Record Matters
It would be easy to treat this history as a list of curiosities to be memorised. It is more useful to read it as evidence of a pattern that the rest of this book will follow.
In every case, a raw material of limited value — milk that would spoil in a day, grain that could not be eaten raw, waste that would otherwise rot in the open — was converted by a living agent into something more valuable, more digestible or safer. In every case the producers controlled the conditions, even without understanding them: the right warmth, the right container, a starter from the last successful batch.
What modern biotechnology added was not the idea. It was knowledge of the agent, and therefore control that is reliable rather than lucky. The chapters ahead are largely the story of how that control was won.
1.3 Milestones in the Growth of Biotechnology
For thousands of years, biotechnology advanced by trial and error. It began to advance by understanding only when two things became possible: seeing the organisms responsible, and explaining how their characters were passed on. Everything in this section follows from those two abilities.
The names below are not a list to be memorised for its own sake. Each one removed a specific obstacle, and each opened the door to the technique that came after it — which is why examiners are fond of asking what would have been impossible without a given discovery.
Seeing the Invisible World
Robert Hooke (1665) examined a thin slice of cork under a compound microscope of his own design and saw that it was built of tiny empty compartments, like the cells of a honeycomb. He named them cells — the word we still use. What Hooke actually saw were the dead cell walls left behind in cork, but the idea that living material is made of units was now on the table.
Living cells were seen for the first time by Anton van Leeuwenhoek (1632–1723), who ground his own lenses and built simple microscopes far more powerful than anything then available. Through them he observed moving, living organisms in pond water, saliva and other samples — the first sight of the microbial world.
This mattered enormously for biotechnology. Until someone could look into a fermenting vat and see what was living in it, there was no way to find out which organism was doing the work, and therefore no way to control it. Leeuwenhoek supplied the instrument; two centuries later that instrument would be turned on a spoiled distillery vat, as the next section describes.
Diagram
Fig. 1.4 – Two panels side by side: (a) Hooke’s drawing of cork showing rows of empty box-like compartments, labelled “cell walls of dead cork cells”; (b) a simple Leeuwenhoek-type microscope with its single bead lens, specimen pin and focusing screw, all labelled
Explaining Inheritance
Gregor Johann Mendel is known as the Father of Genetics for his work on inheritance. Through a series of carefully planned experiments carried out between 1856 and 1863 on pea plants (Pisum sativum), Mendel showed that traits are controlled by discrete units — he called them factors — that are passed unchanged from parents to offspring.
From these experiments he established three principles. The Law of Dominance showed that when two contrasting factors are present, one may mask the expression of the other. The Law of Segregation stated that the two factors of a pair separate from each other during gamete formation, so that each gamete carries only one of them. The Law of Independent Assortment explained that different pairs of factors are distributed to gametes independently of one another.
Mendel’s work laid the foundation of classical genetics and gave heredity a scientific explanation for the first time. Every later development in genetics and biotechnology rests on it, because the whole idea of transferring a desirable character deliberately assumes that characters are carried by units that can be tracked, separated and recombined.
The Modern Era Begins
The twentieth century turned biology into a technology. Three developments did most of the work: the discovery of antibiotics, the working out of the structure of DNA, and the invention of a way to cut and rejoin DNA at will.
Fleming and the Antibiotic Era
Alexander Fleming made a historic contribution to medical science in 1928 with the discovery of penicillin, the first antibiotic. While working with Staphylococcus bacteria, Fleming noticed that a culture plate had been contaminated by a mould, Penicillium notatum, and that the bacterial growth immediately around the mould had been cleared. He concluded that the mould was releasing a substance capable of killing or suppressing bacteria, and he named that substance penicillin.
The discovery marked the beginning of the antibiotic era. Infections that had regularly been fatal — pneumonia, syphilis, infected wounds — became treatable, and mortality from them fell sharply. Fleming’s work laid the foundation of modern antimicrobial therapy, and in 1945 he shared the Nobel Prize in Physiology or Medicine with Howard Florey and Ernst Chain, who had solved the far harder problem of purifying penicillin and producing it in quantity.
That last point is a biotechnological lesson in itself. A useful organism in a dish is a discovery; a useful product in a vial is an industry. Turning one into the other needed large-scale culture of the mould, control of its growth conditions, and recovery of the product from the broth — the same three problems every fermentation industry faces, and the subject of much of this chapter.
The Structure of DNA
Rosalind Franklin and Maurice Wilkins made the experimental contributions that made the discovery of DNA structure possible. Franklin used X-ray diffraction, also called X-ray crystallography, to study DNA fibres. In this technique a beam of X-rays is passed through the sample and the pattern in which it is scattered is recorded; from that pattern the arrangement of atoms can be worked out. Her results clearly indicated a helical structure of uniform diameter, and gave key measurements of the spacing of the strands and the position of the nitrogenous bases. Wilkins also carried out X-ray diffraction studies on DNA fibres that supported the helical model.
Using this experimental evidence, James Watson and Francis Crick proposed the double helix model of DNA in 1953. They showed that DNA consists of two antiparallel polynucleotide strands coiled around each other and held together by specific base pairing between the purines (adenine and guanine) and the pyrimidines (cytosine and thymine). Adenine pairs with thymine through two hydrogen bonds, while guanine pairs with cytosine through three. This specific pairing accounts for the equivalence rule given by Chargaff, that in any sample of DNA the total number of purine bases equals the total number of pyrimidine bases.
The model explained, in one stroke, how DNA could store genetic information, replicate accurately, and transmit hereditary traits from one generation to the next. It supplied the molecular basis of heredity and became the foundation of molecular genetics and modern biotechnology. Watson, Crick and Wilkins were awarded the Nobel Prize in Physiology or Medicine in 1962; Franklin had died in 1958, and the prize is not awarded posthumously. The structure and functions of DNA are studied in detail in Class XII.
Cutting and Joining DNA
Werner Arber and Hamilton O. Smith made a fundamental contribution to molecular biology through the discovery and characterisation of restriction enzymes, which became the essential tools of genetic engineering. These enzymes recognise short, specific sequences in DNA — called restriction sites — and cut the molecule there. Because they cut DNA at defined positions rather than at random, they are also known as molecular scissors. Restriction endonucleases are studied in detail in Class XII.
Herbert W. Boyer and Stanley N. Cohen made a landmark contribution in 1973 by developing the first successful method of recombinant DNA technology, which laid the foundation of modern genetic engineering. They demonstrated that DNA fragments from different biological sources could be cut, joined and replicated inside a living organism. Using restriction endonucleases, Boyer cut DNA at specific sequences, while Cohen used bacterial plasmids — small circular DNA molecules that a bacterium carries in addition to its main chromosome — as vectors to carry these foreign fragments. The fragments were joined to the plasmid DNA with the help of DNA ligase, forming recombinant DNA molecules, and these recombinant plasmids were introduced into Escherichia coli by the process of transformation, in which a bacterium takes up DNA from its surroundings. The bacteria not only accepted the foreign DNA but replicated it along with their own genetic material. Recombinant DNA technology is studied in Class XII.
Flowchart
FC 1.2 – The three abilities that made genetic engineering possible, in the order they were won: see the cell (Hooke, Leeuwenhoek) → explain inheritance (Mendel) → identify the molecule and its structure (Franklin, Wilkins, Watson, Crick) → cut it at a chosen point (Arber, Smith) → join it and multiply it in a host (Boyer, Cohen)
A Timeline of Important Events
The table below gathers the events discussed above with other landmarks, so that you can see the pace at which the subject moved. Notice how the gaps shorten: two centuries separate Hooke from Mendel, but barely two decades separate the double helix from the first recombinant DNA molecule.
| Period | Event |
|---|---|
| c. 8000 BC | Domestication of animals and cultivation of crops begins |
| c. 6000 BC | Wine and beer are produced by fermentation |
| c. 4000 BC | Bread and curd are made; leavened bread is baked in Egypt |
| 1665 | Hooke observes and names cells in cork |
| 1670s | Leeuwenhoek observes living microorganisms |
| 1761 | Successful cross-breeding of crops is accomplished |
| 1797 | The first child is inoculated with a viral vaccine against smallpox |
| 1830–1833 | The first protein is described and the first enzyme is isolated |
| 1835–1855 | Schleiden and Schwann state that all organisms are composed of cells and that every cell arises from a pre-existing cell |
| 1857 | Pasteur shows that microbes cause fermentation |
| 1859 | Darwin publishes his theory of evolution |
| 1865 | Mendel reports his work on garden peas |
| 1870–1890 | Cotton varieties are improved by crossbreeding; staining methods for identifying bacteria are developed; the first centrifuge is built; chromatin is discovered |
| 1906 | The term genetics is used for the first time |
| 1914 | Bacteria are first used to treat sewage, in England |
| 1919 | The word biotechnology first appears in print |
| 1928 | Penicillin is discovered as an antibiotic |
| 1941 | The term genetic engineering is first used |
| 1944 | DNA is proved to carry genetic information |
| 1953 | The double helix model of DNA is proposed |
| 1961 | The first biopesticide is registered |
| 1966 | The genetic code is worked out |
| 1973 | The first recombinant DNA molecules are made and cloned in bacteria |
| 1977 | Human genes are expressed in bacteria for the first time |
| 1979 | Human growth hormone is produced by recombinant means |
| 1983 | The polymerase chain reaction is devised |
| 1984 | DNA fingerprinting is developed |
| 1986 | Interferon is released as the first anti-cancer drug of its kind |
| 1988 | A patent is granted for the first genetically altered animal, a mouse |
| 1990 | The first transgenic dairy cow is created to produce human milk proteins for infant formula |
| 1994 | The first gene associated with breast cancer is identified |
| 1996–1997 | Dolly the sheep, the first mammal cloned from an adult cell, is born and announced |
| 2002 | A vaccine against cervical cancer is reported |
One major figure has been deliberately left out of this account. Louis Pasteur’s contribution was so central — and so directly connected to fermentation, the subject of the rest of this chapter — that it is taken up on its own in the next section.
1.4 Louis Pasteur and the Beginnings of Modern Biotechnology
Louis Pasteur, who began the pasteurization of milk and identified the role of microorganisms in fermentation, is regarded as the Father of Modern Biotechnology. The chapter opened with the problem that started him off: a distillery at Lille whose vats were turning sour, and a professor who chose to look at the fermenting liquid through a microscope. This section follows what came out of that investigation.
Pasteur’s work on the Lille problem grew into a long series of studies on alcoholic fermentation. He went on to investigate fermentation broadly, including the production of the lactic acid responsible for the souring of milk, and he also studied butyric acid fermentation. In each case the same pattern appeared: a particular change in the liquid went with the presence of a particular kind of organism.
The Germ Theory of Fermentation
In 1857 Pasteur presented experimental evidence that living organisms take part in all fermentative processes, and that a specific organism is associated with each particular fermentation. This evidence gave rise to the germ theory of fermentation.
The claim was more radical than it now sounds. Leading chemists of the day held that fermentation was a purely chemical decomposition of sugar, and that the yeast seen in the liquid was a by-product of the change rather than its cause. Pasteur reversed the order: the organism was not a consequence of fermentation but the agent of it. He also showed, in related experiments, that a sterile nutrient broth protected from airborne organisms does not ferment or putrefy on its own — fermentation does not arise spontaneously, but has to be seeded by living cells.
Once each fermentation had a named organism behind it, industrial fermentation stopped being a gamble. A brewer could now aim to keep one organism alive and all others out, which is exactly what a modern fermenter is designed to do.
The Pasteur Effect
Pasteur’s studies on fermentation led him to an unexpected finding. He observed that fermentation could be arrested by passing air through the fermenting fluid. He concluded that this happened because the organism responsible could function only in the absence of oxygen — and this led him to introduce the terms aerobic and anaerobic to describe organisms that live in the presence and in the absence of oxygen respectively.
In 1861 Pasteur went further. He suggested that in the absence of oxygen — that is, under anaerobic respiration — yeast consumes far more glucose than it does in the presence of oxygen. The reason is that the energy released per molecule of glucose is much less anaerobically, about 2 ATP, compared with about 38 ATP when oxygen is available. A cell that needs a fixed amount of energy must therefore burn through a great deal more sugar when it is deprived of oxygen. This phenomenon is known as the Pasteur Effect.
The Pasteur Effect has a direct industrial use, and it explains a step that often puzzles students of alcohol production. A brewer or distiller wants two different things from the same organism at two different times: first a large, healthy population of yeast, and then ethanol from that population. The first is achieved by culturing the yeast in an aerated broth, where oxygen allows it to grow rapidly and multiply. Air is then shut out, the yeast switches to fermentation, and the sugar is converted to alcohol. The same effect is used in reverse in bread-making, where a well-aerated dough favours yeast growth and gas production rather than alcohol.
Flowchart
FC 1.3 – Two-stage use of yeast in alcohol production: yeast inoculum → aerated broth, oxygen supplied → rapid multiplication, high yeast biomass → air excluded, vessel sealed → anaerobic fermentation of sugar → ethanol and carbon dioxide
Pasteurization
Pasteur applied his knowledge of microbes and fermentation to the wine and beer industries. In 1863 he studied the contamination of wine and showed that it was caused by microorganisms. To prevent it he used a simple procedure: he heated the wine to 60 °C and then cooled it down immediately to a very low temperature. The process is now known universally as pasteurization, and today it is applied to many foods and beverages, particularly milk.
Two details of the procedure are worth thinking about rather than memorising. The first is why the temperature is kept moderate at all: boiling would kill more organisms, but it would also change the flavour of wine and the taste and some vitamin content of milk, and the product would no longer be what the consumer wanted. Pasteurization is a compromise, deliberately chosen.
The second is why cooling must follow immediately. Pasteurization reduces the microbial population sharply but does not sterilise the product — a few heat-resistant organisms and spores survive. If the warm liquid were left standing, those survivors would find an ideal temperature and a food supply with no competitors left, and would multiply rapidly. Fast cooling denies them that opportunity.
Pasteur and Beer
After his success with wine, Pasteur turned his attention to beer. By developing practical techniques for the control of beer fermentation — keeping the fermenting liquid free of unwanted organisms and holding conditions steady — he was able to devise a method of manufacture that prevented the product from deteriorating over long periods.
This is the point at which Pasteur’s science joins the industry described later in this chapter. The malting, mashing, fermentation and maturation steps of modern beer production in Section 1.8 are all attempts to answer the question Pasteur first posed at Lille: which organism is at work in this vessel, and how do I make sure it is the only one?
1.5 Traditional and Modern Biotechnology
Everything described so far belongs to one subject, but not to one method. The curd-setter of four thousand years ago and the scientist who moves a gene from one organism into another are both practising biotechnology, and both are upgrading a raw material biologically. What separates them is the level at which they work.
It is usual, therefore, to divide the techniques of biotechnology into two groups: traditional (also called conventional or old) biotechnology, and modern biotechnology. The division is not a division of importance — traditional techniques still feed and clothe most of the world — but of the tools available and the degree of control they give.
1.5.1 Traditional Biotechnology
Traditional biotechnology uses whole living organisms, or naturally occurring populations of them, to carry out a useful change, without any deliberate alteration of their genetic material.
Its characteristic features follow from that definition. The organism is used as it is found, so the product is limited to what the organism can already make. The methods are largely empirical — worked out by trial, error and long experience rather than from theory. Conditions are controlled indirectly, by warmth, timing, containers and a starter from a previous batch. And because a natural population is being used, results vary from batch to batch.
Its applications are the familiar ones. Fermented foods and beverages — curd, cheese, buttermilk, idli and dosa batter, bread, vinegar, wine and beer — are all traditional biotechnology. Selective breeding of crop plants and livestock, described in Section 1.2, improves inherited characters by choosing parents rather than by altering genes. Waste management by composting and by microbial treatment of sewage uses naturally occurring soil and water organisms to break down material that would otherwise accumulate. Silage-making, in which green fodder is preserved for cattle by allowing lactic acid bacteria to acidify it, preserves feed through a dry season by exactly the mechanism that preserves milk as curd.
1.5.2 Modern Biotechnology
Modern biotechnology works below the level of the whole organism — at the level of the cell, the chromosome and the gene. Its defining ability is that a specific gene can be identified, isolated, and transferred into another organism, which then makes a product it could never have made naturally.
Three features distinguish it. It is specific: one chosen character is transferred, rather than the whole set of characters that a cross between two parents would bring. It crosses species barriers: a gene from a bacterium can be placed in a plant, which no amount of breeding could achieve. And it is fast: a result that selective breeding might reach over many generations can be obtained in a single step.
Its main technique groups are named here only; each is studied in detail in Class XII.
- Recombinant DNA technology — cutting DNA at chosen sites, joining a chosen fragment into a vector, and multiplying it inside a host cell.
- Cell and tissue culture — growing cells, tissues or whole organisms under sterile conditions in a defined nutrient medium.
- Gene analysis techniques — methods that identify, copy and read DNA sequences, used in diagnosis and in forensic work.
- Hybridoma technology and immunological techniques — used to obtain antibodies of a single defined specificity for diagnosis and treatment.
The applications are correspondingly wide. In agriculture, Bt cotton carries a bacterial gene that makes the plant resistant to certain insect pests, and Golden Rice was developed to accumulate a precursor of vitamin A in the grain. In healthcare, the recombinant hepatitis-B vaccine is produced by yeast carrying a single viral gene, so that the vaccine contains no virus at all. In the environment, bioremediation uses selected microorganisms to break down oil spills and industrial effluents. In horticulture and forestry, micropropagation raises thousands of identical, disease-free plants from a small piece of tissue.
Flowchart
FC 1.4 – How control has tightened across the two phases: unknown agent, uncontrolled outcome (ancient fermentation) → known agent, controlled conditions (Pasteur onwards; traditional biotechnology practised scientifically) → chosen gene in a chosen host, defined product (modern biotechnology)
The Two Are Not Rivals
It is a common mistake to read this comparison as though modern biotechnology has replaced traditional biotechnology. It has not. Far more people eat fermented food every day than take a recombinant medicine, and the fermentation industries remain among the largest users of biological processes in the world.
What has happened is that the two have merged in practice. An industrial fermenter today may well be running a traditional process — the conversion of sugar to a useful product by a microorganism — using a strain that has been improved by modern techniques, under conditions monitored by instruments Pasteur would have envied. The organism is modern; the biochemistry is ancient.
1.6 The Kitchen as the First Biotechnology Laboratory
Biotechnology begins at home, in the kitchen. Ever since people began to cook their food and keep a place to prepare it in, they have been developing ways of making fermented products in that same room — and doing it, for most of history, without any idea of what was actually carrying out the change.
Look at what a kitchen routinely produces. Milk is converted into curd, yoghurt, buttermilk, srikhand and paneer. Wheat and pulses are turned into bread, cake, dosa, idli, bhatura, naan and dhokla. In each case a raw material is left under chosen conditions until an organism has changed it into something better to eat and longer-lasting than what went in. That is the whole definition of biotechnology, carried out with a vessel, a warm corner and a spoonful of yesterday’s batch.
Why the Kitchen Counts as a Laboratory
The claim is more than a figure of speech. Compare a kitchen with any laboratory in this book and the same four elements are present.
A biological agent is chosen and supplied — a spoonful of old curd, a pinch of yeast, or the natural organisms already present on soaked grain. A substrate is provided — milk, flour, batter. Conditions are controlled: the milk is boiled first and then cooled to the right warmth, the vessel is covered, the pot is left in a warm place for a set number of hours. And a defined product is expected, with the cook judging success by texture, smell and taste rather than by an instrument.
What the kitchen lacks is not method but measurement, and not control but explanation.
1.6.1 The Reasoning Behind Curd
The production of curd from milk is the classic example — a process humans have practised for thousands of years without any formal understanding of microbiology.
In this process a small amount of starter culture, which is simply a little curd from a previous batch, is added to warm milk. The starter contains lactic acid bacteria (LAB), mainly species of Lactobacillus, and these act as the biological agents that perform the fermentation.
These bacteria convert lactose, the sugar naturally present in milk, into lactic acid. As lactic acid accumulates, the pH of the milk falls. The milk protein casein is stable at the pH of fresh milk but not in acid; as the pH drops, casein loses its stability, coagulates, and traps the liquid around it, so that the milk sets into a thick, semi-solid product — curd.
This single biochemical transformation does several useful things at once. It improves the digestibility, taste, texture and nutritional value of milk. It increases the vitamin B content of the product. And because the environment is now acidic, it inhibits the growth of harmful microorganisms, so that the curd keeps longer than the milk it was made from and is safer to eat.
Flowchart
FC 1.5 – Three routes to setting curd, all converging on the same step. Route 1: 1 litre fresh milk → boil to 100 °C → cool to 37 °C. Route 2: cold pasteurised milk → warm to 37 °C. Route 3: refrigerated boiled milk → warm to 37 °C. All three → add a teaspoonful of curd as starter → leave at room temperature for 4 hours → curd → leave for a further 2 hours → two-phase separation into curd and whey
Notice what the three routes in FC 1.5 have in common. Whichever milk you begin with, it must first be brought to a state in which competing organisms are few and the temperature is right. Fresh milk needs boiling and cooling; pasteurised and refrigerated boiled milk have already been heat-treated, so they need only warming. The step that cannot be skipped in any route is bringing the milk to about 37 °C before the starter goes in.
Diagram
Fig. 1.5 – A set curd left standing, shown in section: the firm white curd mass below and the clear yellowish whey layer separated above, labelled “two-phase separation”; alongside, a simple representation of casein particles dispersed in fresh milk and the same particles clumped together after the pH has fallen
In your practical work you will test this reasoning directly. One of the ISC Class XI experiments asks you to study the effect of temperature on the curdling of milk by Lactobacillus at 37 °C, 60 °C and 10 °C — and the results follow from what has just been said: the bacteria are in their active form at 37 °C, are inactive at low temperature, and are killed at high temperature.
1.6.2 Other Kitchen Fermentations
Curd is not an isolated case. Kitchens across India produce a wide range of fermented foods, each using a different substrate and each yielding a different texture and taste. The table below lists some of them.
| Product | Substrate | Nature and use |
|---|---|---|
| Idli | Rice and black gram | Steamed, spongy cake; breakfast food |
| Dosa | Rice and black gram | Spongy pan cake, shallow-fried; staple food |
| Papad | Black gram | Circular wafers; snack |
| Wari | Black gram | Spongy cake; snack |
| Bhatura | Wheat | Flat deep-fried, leavened bread; snack |
| Naan | Wheat | Leavened flat baked bread; staple food |
| Jalebi | Wheat | Crispy, deep-fried sweet confectionery |
| Dahi | Milk | Thick gel; savoury |
| Dhokla | Bengal gram | Spongy cake; snack |
| Khaman | Bengal gram | Spongy cake; breakfast food |
| Rabadi | Wheat, pearl-millet or barley with buttermilk | Cooked paste; staple food |
| Srikhand | Milk | Concentrated, sweetened, savoury |
| Mishti dahi | Milk | Thick gel; sweet savoury |
| Gundruk | Leafy vegetable | Sun-dried, sour-acidic taste; soup or pickle |
| Sinki | Radish tap root | Sun-dried, sour-acidic taste; soup or pickle |
| Mesu | Bamboo shoot | Sour-acidic; pickle |
Two patterns are worth extracting from this list rather than memorising it. The cereal and pulse batters — idli, dosa, dhokla, khaman — are left to stand until the natural organisms on the soaked grain produce acid and gas; the gas is what makes the steamed or fried product spongy. The vegetable preparations — gundruk, sinki, mesu — are acidified by lactic acid bacteria and then dried, so that both the acidity and the loss of water work together to preserve a vegetable through a season when nothing is growing.
Wheat products such as naan and bhatura depend on a different organism. Here yeast ferments the sugars in the dough, releasing carbon dioxide that is trapped by the dough and makes it rise, giving the baked bread its light, open texture.
What the Kitchen Demonstrates
Curd formation in the kitchen shows the fundamental principles of biotechnology in miniature — the use of living organisms, the control of conditions, and a biochemical reaction that yields a useful product. Long before modern laboratories and industrial fermenters existed, such practices show how human beings were applying biotechnology in daily life without knowing it, which is what makes the kitchen the earliest and most accessible biotechnology laboratory.
The step from here to industry is a step of scale and instrumentation, not of principle. In the sections that follow, the same three requirements reappear — the right organism, the right substrate, the right conditions — but now in vessels holding thousands of litres, with the temperature, pH and oxygen supply held steady by design instead of by habit.
1.7 Fermentation and Its Types
Every product discussed so far — curd, bread, wine, beer, vinegar, silage — comes from the same underlying process. It is time to name that process properly, because the whole of the fermentation industry, and the beer production described in the next section, rests on it.
Fermentation is a form of anaerobic respiration: carbon dioxide may be produced, but no atmospheric oxygen is used. This is why it is described as respiration without air. In it, an organic substrate is broken down incompletely, energy is released in small amount, and organic end products are left behind.
Fermentation is the principal way of obtaining energy for organisms living where oxygen is scarce, and many yeasts and bacteria can switch to it when air is excluded. It is usually distinguished from anaerobic respiration in general by the position of the substrate: in fermentation the substrate lies outside the cell in soluble form, and the organism acts on it from within its surroundings.
Why Fermentation Suits Industry
Two features of fermentation make it valuable to industry, and both follow from the fact that the breakdown is incomplete.
First, the end products are useful. If a sugar were oxidised completely, nothing would remain but carbon dioxide and water. Because fermentation stops part-way, the half-used molecule that remains — ethanol, lactic acid, acetic acid — is itself the product that is wanted.
Second, the process needs no oxygen supply. Keeping thousands of litres of broth properly aerated is expensive and difficult; a sealed anaerobic vessel is simpler and cheaper to run.
Under the specific conditions provided in a laboratory or an industry, plant cells and plant-associated microorganisms obtain energy through fermentation pathways, and it is these pathways that have industrial relevance. On the basis of the nature of the end products formed, fermentation is classified into homofermentation and heterofermentation.
1.7.1 Homofermentation
Homofermentation is a type of anaerobic respiration in which glucose is broken down into a single main end product, usually lactic acid, with the release of energy. No carbon dioxide is produced in this process. It is commonly seen in lactic acid bacteria such as Lactobacillus, and it is important in the formation of curd, yoghurt and other fermented foods.
\[\text{C}_6\text{H}_{12}\text{O}_6 \longrightarrow 2\,\text{C}_3\text{H}_6\text{O}_3 + 2\,\text{ATP}\]
Here \(\text{C}_6\text{H}_{12}\text{O}_6\) is glucose and \(\text{C}_3\text{H}_6\text{O}_3\) is lactic acid; ATP is adenosine triphosphate, the molecule in which the cell stores the energy released.
Steps involved
- Glycolysis: glucose is converted to 2 molecules of pyruvate, yielding 2 ATP and 2 NADH.
- Reduction of pyruvate: pyruvate combines with NADH to give lactic acid and NAD⁺, the reaction being catalysed by the enzyme lactate dehydrogenase.
The second step deserves a moment’s thought, because it explains why fermentation exists at all. Glycolysis can continue only while a supply of NAD⁺ is available to accept hydrogen. In the presence of oxygen, NADH is re-oxidised in the mitochondria. Without oxygen that route is closed, and the cell would grind to a halt after a few reactions. Reducing pyruvate to lactic acid regenerates NAD⁺ and keeps glycolysis running. The lactic acid is, from the organism’s point of view, simply a place to dump hydrogen — and from ours, the product.
1.7.2 Lactic Acid Fermentation
Lactic acid fermentation is the form of anaerobic respiration in which lactic acid bacteria (LAB) convert lactose, the milk sugar, into lactic acid. The process is widely used in the dairy and probiotic industries because it improves the taste, texture, digestibility, shelf life and health benefits of milk products.
In the dairy industry, lactic acid fermentation is central to the production of curd, yoghurt, cheese, buttermilk and fermented milk drinks. Section 1.6 followed the sequence in detail — the starter culture, the fall in pH, the coagulation of casein — and the industrial process is the same sequence carried out in a controlled vessel, with a defined starter strain rather than a spoonful of the previous batch. Besides preserving the product naturally, the process enhances flavour, aroma and nutritional quality, particularly by increasing the B-complex vitamin content and improving mineral absorption.
In the probiotic industry, lactic acid fermentation is used to produce foods and supplements containing beneficial live microorganisms that promote gut health. Probiotic LAB help maintain a healthy intestinal microflora, improve digestion, enhance immunity and prevent gastrointestinal infections. During fermentation these bacteria produce lactic acid together with other bioactive compounds that suppress harmful microbes in the gut. Probiotic yoghurt, fermented milk drinks and capsules are manufactured by carefully controlled lactic acid fermentation, so that the beneficial bacteria remain alive and effective in the finished product.
That last requirement is the difficult one. The organism in a probiotic product is not merely a processing agent to be discarded, as yeast is in beer; it is the product itself, and it must survive manufacture, packaging, storage and the acid of the stomach. This is why probiotic manufacture depends heavily on strain selection and on strict control of temperature and time.
Lactic acid fermentation thus plays a crucial role in both the dairy and the probiotic industries, combining food technology, microbiology and biotechnology to produce nutritious, safe and health-promoting products.
1.7.3 Heterofermentation
Heterofermentation is a type of anaerobic respiration in which glucose is converted into more than one end product — mainly lactic acid, ethanol and carbon dioxide — along with the release of energy. It occurs in certain bacteria and yeasts and is significant in the bakery, brewing and alcohol industries.
\[\text{C}_6\text{H}_{12}\text{O}_6 \longrightarrow \text{C}_2\text{H}_5\text{OH} + \text{C}_3\text{H}_6\text{O}_3 + \text{CO}_2 + \text{ATP}\]
where \(\text{C}_2\text{H}_5\text{OH}\) is ethanol and \(\text{CO}_2\) is carbon dioxide.
Steps involved
- Glucose enters a metabolic pathway called the pentose phosphate pathway.
- Lactic acid, ethanol and \(\text{CO}_2\) are formed.
- NAD⁺ is regenerated so that the breakdown of glucose can continue.
- The end products are ethanol, lactic acid and carbon dioxide.
- Only 1 ATP is released.
The lower energy yield is the price of the different route. A homofermentative organism runs glucose through glycolysis and collects 2 ATP; a heterofermentative organism takes a pathway that releases carbon as \(\text{CO}_2\) along the way and collects only 1. In return it produces a mixture of products — and it is that mixture, particularly the gas, that the baker wants.
1.7.4 Alcoholic Fermentation
Alcoholic fermentation is an anaerobic metabolic process in which simple sugars are converted into ethanol and carbon dioxide, with the release of a small amount of energy. It is carried out mainly by yeast, especially Saccharomyces cerevisiae, and it is of great importance in the bioethanol industry and the beverage industry.
\[\text{C}_6\text{H}_{12}\text{O}_6 \longrightarrow 2\,\text{C}_2\text{H}_5\text{OH} + 2\,\text{CO}_2 + 2\,\text{ATP}\]
In the bioethanol industry, alcoholic fermentation is used to produce ethanol as a renewable biofuel. Plant-based raw materials such as sugarcane, maize, molasses and starch-rich crops are first broken down into fermentable sugars. Yeast then ferments these sugars under anaerobic conditions to ethanol and carbon dioxide. The ethanol obtained is purified by distillation and used as a fuel or fuel additive, which helps reduce dependence on fossil fuels and lowers greenhouse gas emissions.
The reason a starch-rich crop must be “first broken down” is worth noting, because the same requirement returns in the next section. Yeast cannot ferment starch. Starch is a polysaccharide, and the yeast has no means of splitting it; the raw material must first be hydrolysed to simple sugars by enzymes before fermentation can begin at all.
In the beverage industry, alcoholic fermentation is responsible for the production of beer, wine and other alcoholic drinks. In wine production, the sugars present in fruit juices, especially grape juice, are fermented by yeast to ethanol, which gives the beverage its alcohol content, and to carbon dioxide, which may contribute effervescence. In beer production, sugars derived from malted grains undergo fermentation. Alcoholic fermentation also contributes to the flavour, aroma and quality of alcoholic beverages.
Alcoholic fermentation is thus a key industrial process linking plant-derived sugars, microbial metabolism and biotechnology in the production of biofuels and alcoholic beverages.
Flowchart
FC 1.6 – Classification of fermentation by end product: fermentation of sugar (anaerobic) → branch 1, homofermentation → single end product, lactic acid → dairy and probiotic industries; branch 2, heterofermentation → lactic acid + ethanol + \(\text{CO}_2\) → bakery, brewing and alcohol industries; branch 3, alcoholic fermentation by yeast → ethanol + \(\text{CO}_2\) → beverage and bioethanol industries
1.8 Large-Scale Production of Beer
The kitchen showed the principle of fermentation; beer production shows what happens when that principle is scaled up into an industry. It is worth studying closely, because every problem an industrial fermentation must solve appears here in a form you can follow from grain to bottle.
Beer is an undistilled alcoholic beverage in which the alcohol content is quite low, ranging between 4 and 8 per cent. It is produced by the fermentation of barley or other cereals, using Saccharomyces cerevisiae. To enhance the fermentation, a few adjuncts such as wheat and maize, which are rich in starch, are also added.
The Problem the Process Must Solve
Before following the steps, notice the difficulty. Yeast ferments simple sugars, but barley grain stores its food as starch, a polysaccharide the yeast cannot use. Adding yeast to crushed barley and water would achieve almost nothing.
The starch must therefore be broken down to fermentable sugars before the yeast is introduced — and the neatest way to do that is to let the barley grain supply the enzymes itself. A germinating seed must mobilise its own starch reserves to grow, and to do so it produces starch-digesting enzymes. The brewer’s trick is to start the seed germinating, let it make those enzymes, and then stop it before it consumes the very starch that the process needs.
That trick is the first step, and it explains the whole shape of what follows.
Flowchart
FC 1.7 – Beer production: barley grain → malting (cleaning, soaking 2 days, germination 4–6 days, heating to about 80 °C, powdering) → malt → mashing with hot water at 55–65 °C, starch hydrolysed to sugars, hops added → beer wort → fermentation by Saccharomyces cerevisiae at 20–28 °C for 5–10 days, anaerobic → green beer → maturation at 0–3 °C for a few weeks, followed by chill-proofing → carbonation with \(\text{CO}_2\) → bottling or canning → pasteurization → finished beer
1.8.1 Malting
Dried barley is cleaned and soaked in water for a period of two days. The excess water is then removed and the grain is incubated for germination for four to six days.
The process of germination forms the enzymes amylase and protease, for the hydrolysis of starch and protein respectively. The temperature is then increased to about 80 °C to stop germination, but the enzymes remain in the active form. Malt is then prepared by powdering the seeds.
1.8.2 Mashing
The powdered malt is mixed with hot water at 55–65 °C. This mashing of the malt separates the soluble materials and the supplementary ingredients from the malt.
It also leads to the degradation of starch by amylase, producing maltose, dextrin and glucose, and of proteins by proteases, producing peptones, peptides and amino acids. The end of mashing results in a medium suitable for fermentation, called beer wort.
The temperature range is not arbitrary. Enzymes work fastest at their optimum temperature and are destroyed above it, so 55–65 °C is chosen as the band in which the malt amylases act rapidly without being denatured. Take the mash much hotter and the enzymes are lost before their work is done; keep it much cooler and conversion is too slow to be practical.
The products of mashing matter to the finished beer in two different ways. The sugars are the yeast’s food and become alcohol. The peptides and amino acids are largely left behind, and they contribute to the body, foam and nutritional value of the beer — which is also why some of them must be removed later, as you will see.
The dried female flowers of the hop plant are added at this stage. They provide flavour, a specific aroma, and a stabilising effect on the beer.
1.8.3 Fermentation
The beer wort is now inoculated with yeast in open or closed fermentors. Fermentation is carried out at a temperature of 20–28 °C for 5 to 10 days, and is achieved by the use of the yeast Saccharomyces cerevisiae.
Under anaerobic conditions the yeast carries out alcoholic fermentation, converting the sugars of the wort into ethanol and carbon dioxide by the reaction already given in Section 1.7.
Everything about this stage is a controlled version of what a fermenting jar does badly. The temperature is held in a narrow band because yeast activity, and the balance of flavour compounds it produces, both depend on it. The vessel excludes air so that the Pasteur Effect works in the brewer’s favour and the sugar goes to alcohol rather than to more yeast. And the wort was sterile and hop-protected when the yeast went in, so that the organism inoculated is the only one working.
Diagram
Fig. 1.6 – A closed cylindrical fermentor for beer, shown in section and labelled: wort inlet, yeast inoculation port, cooling jacket with coolant in and out, temperature probe, carbon dioxide outlet through a one-way valve, sampling valve, and the yeast sediment collecting at the conical base
1.8.4 Maturation
The fermented fluid is transferred to storage tanks maintained at a temperature range of 0–3 °C. The storage period is roughly a few weeks, during which sedimentation of yeast cells and precipitation of nitrogenous substances, resins, phosphates and similar materials occurs.
The partially matured beer is then subjected to chill-proofing, which involves the removal of residual proteins. These proteins are the reason freshly fermented beer is turbid; unless they are removed, the beer would turn cloudy every time it was chilled in the consumer’s refrigerator.
Cold storage does two jobs at once here. It slows the yeast almost to a stop, ending fermentation at the desired alcohol content, and it makes the unwanted proteins and resins less soluble so that they settle out of their own accord instead of having to be extracted.
1.8.5 Carbonation
Carbonation of beer is usually carried out by injecting carbon dioxide. The matured and carbonated beer is then bottled or canned, and pasteurized at 60 °C for about 20 minutes.
Since the fermentation itself produced carbon dioxide, it may seem strange to add more. The gas produced during fermentation is largely lost during the long cold maturation, and the amount remaining is neither sufficient nor consistent from batch to batch. Injecting a measured quantity gives every bottle the same degree of effervescence — a matter of quality control rather than of chemistry.
The final pasteurization step is the one already met in Section 1.4, applied here to a packaged product: it destroys surviving yeast and spoilage organisms so that the beer does not continue to change in the bottle.
The technological details of malt preparation and of the brewing industry are among the project topics suggested in the ISC syllabus, and a visit to a fermentation unit — or a careful study of its published process flow — is the easiest way to see how the five steps above are laid out as real vessels and pipework.
1.9 Good Laboratory Practices
A biotechnological product begins as a set of experimental results. Before a vaccine, an enzyme or a diagnostic kit can be considered for use, someone must be able to trust the data that says it is effective and safe — and that trust cannot rest on the reputation of the scientist alone. It has to rest on the way the work was done and recorded.
Good Laboratory Practices (GLP) are a set of basic rules and guidelines that must be followed while carrying out research work, routine experiments and trial experiments in laboratories. These guidelines apply to research laboratories as well as to manufacturing and testing establishments.
GLP ensures the safety of laboratory personnel, the public and the environment, and it also guarantees that experimental data are accurate, reliable, reproducible and ethically obtained. It is especially important during product development, because studies relating to health and environmental safety are compulsory before any product meant for human consumption or use is released into the market.
Objectives of GLP
- To ensure accuracy and reliability of experimental data.
- To maintain safety of laboratory workers, the public and the environment.
- To ensure ethical conduct of research and experiments.
- To maintain uniform standards in laboratory testing and research.
The fourth objective is easily overlooked but is the reason GLP exists as an international system rather than a house rule. If every laboratory recorded its work in its own way, results from two countries could not be compared, and a safety study accepted in one place would have to be repeated in another. Uniform standards make data portable.
1.9.1 Principal Components of GLP
GLP rests on four main components, which together ensure quality and safety in laboratory work.
1. Management. Management is the administrative authority responsible for planning, coordinating and supervising laboratory activities. It ensures the proper implementation of GLP guidelines and monitors the functioning of the various safety and ethics committees.
2. Quality Assurance (QA). Quality Assurance is an internal monitoring system or committee that ensures GLP is followed in day-to-day laboratory work. It checks experimental procedures, records and reports. The Animal Ethics Committee and the Bioethics Committee are examples of QA bodies.
3. Study Director. The Study Director is responsible for the overall conduct of research studies and safety experiments. This person ensures that experiments are carried out according to approved procedures, that data are recorded correctly, and that safety rules are strictly followed.
4. National Compliance Monitoring Authority (NCMA). The National Compliance Monitoring Authority is a national regulatory body that approves safety studies relating to the health and environmental impact of new biotechnology products. Approval from this authority is necessary for international recognition and acceptance. The guidelines for GLP are provided by the Organisation for Economic Co-operation and Development (OECD) — the same body whose definition of biotechnology you met in Section 1.1.
Flowchart
FC 1.8 – Lines of responsibility under GLP: Management (plans, resources, appoints committees) → Study Director (conducts the study, records the data) → Quality Assurance, including the Animal Ethics and Bioethics Committees (monitors internally, audits records) → National Compliance Monitoring Authority (inspects and approves externally) → international recognition of the data
Notice the structure in FC 1.8. Three of the four components sit inside the organisation and one sits outside it. Internal monitoring alone would be a laboratory marking its own answer script; external inspection alone would catch problems only after the fact. GLP uses both, which is why its data are accepted across borders.
1.9.2 Basic Laboratory Practices under GLP
- Eating and drinking are strictly prohibited in laboratories.
- Laboratory coats must be worn by all workers.
- Protective equipment such as gloves, fume cupboards, masks and safety shields must be used.
- Proper training and instructions should be followed while using new instruments.
- Laboratories should be kept clean, with proper sewage and drainage.
- Glassware must be properly washed and rinsed.
- Safe disposal of waste — chemical, biological and radioactive — must be ensured.
Each of these rules protects something specific. Rules (a) to (c) protect the worker from the material. Rules (e) and (f) protect the experiment from contamination, since a dirty bench or an inadequately rinsed flask can introduce organisms or residues that quietly ruin a result. Rule (g) protects the world outside the laboratory: a culture of microorganisms poured down a sink is not merely untidy, it is a release into the environment.
Example of GLP. Consider a microbial culture experiment carried out under GLP. The media are sterilised before use, the samples are properly labelled, protective equipment is worn, and the observations are recorded systematically as they are made. Every one of these steps is present in the practical work of this course — you will sterilise media in an autoclave, label the culture plates you prepare, take precautions when handling a needle for a blood sample, and enter your readings in a practical file at the time of the experiment rather than reconstructing them afterwards. The habits GLP asks of a professional laboratory are the habits the practical file is meant to build.
1.10 Quality Control Management and Good Manufacturing Practices
GLP governs how a product is investigated. It says nothing about how that product is then made, thousands of doses at a time, month after month. A vaccine that performed well in a trial is of no use if the batch delivered to a clinic is weaker, contaminated or wrongly labelled.
Two systems close this gap. Quality control management checks the product itself, and Good Manufacturing Practices govern the process that makes it.
1.10.1 Quality Control Management of Products
Quality control (QC) is the part of a manufacturing system in which materials, intermediates and finished products are actually tested against defined standards, and in which a batch is released for sale only if it meets them.
A biological product makes this harder than a chemical one. Living organisms vary; a fermentation batch is never exactly identical to the one before it; and a protein product can lose activity without any visible change. Quality control in biotechnology therefore checks a product in several distinct ways.
Identity — is the product what the label says it is? Purity — is it free of contaminating organisms, host-cell proteins and other unwanted material? Potency — is the biological activity of the required strength, since a vaccine or an enzyme is measured by what it does, not merely by how much of it there is? Safety — is it free of toxins and pyrogens, substances that would cause fever if injected? And stability — will it still meet all of these standards at the end of the shelf life printed on the pack?
Testing only the finished product would be a poor strategy, because by then a whole batch has already been made and any fault is expensive and irreversible. Quality control is therefore applied at every stage, so that a problem is caught where it arises.
Flowchart
FC 1.9 – Quality control across a production run: incoming raw materials and media tested → in-process checks during fermentation (pH, temperature, sterility, cell count, product concentration) → checks during downstream processing and purification → finished batch tested for identity, purity, potency, safety → batch record reviewed against specification → batch released, or rejected and investigated
Two ideas support this whole structure. Sampling allows a defined portion of a batch to be tested and the result taken to represent the whole, since testing every vial would destroy the product. Documentation records what was done and what was found, so that if a fault appears later, the batch can be traced back through its records to the step where it went wrong.
1.10.2 Good Manufacturing Practices (GMP)
Good Manufacturing Practices (GMP) are a set of guidelines and standards followed during the manufacture, processing, packaging and storage of biotechnological products, to ensure that they are consistently produced with high quality, safety and efficacy. GMP is essential in industries producing vaccines, antibiotics, enzymes, hormones and recombinant products.
The word to hold on to is consistently. GMP is not aimed at making one excellent batch; it is aimed at making every batch the same as the approved one. A patient who receives the ten-thousandth dose of a vaccine must receive what the trial subjects received.
Objectives of GMP
- To ensure quality, safety and effectiveness of biotechnological products.
- To prevent contamination, mix-ups and manufacturing errors.
- To maintain uniformity and consistency in large-scale production.
- To protect consumer health and public safety.
Principles of GMP
- Well-designed and hygienic manufacturing facilities with clean-room conditions.
- Trained and qualified personnel following defined responsibilities.
- Use of standard operating procedures (SOPs) for all production steps.
- Regular quality control and quality assurance checks.
- Proper documentation and record keeping at every stage.
- Safe storage, packaging and distribution of finished products.
Each principle answers a specific way in which large-scale production can fail. Clean-room conditions and hygienic design answer contamination — the risk that dominates any process using living cells. Trained personnel with defined responsibilities answer the problem of a task being done differently by different people, or of nobody being accountable for it. Standard operating procedures, written step-by-step instructions for each operation, answer the same problem from the other side: the procedure does not change when the operator does. Documentation makes the whole run traceable afterwards, and controlled storage and distribution protect a product that may lose potency if the cold chain is broken between factory and patient.
Example of GMP. In the production of vaccines in a biotechnology industry, manufacturing is carried out under sterile conditions, equipment is regularly cleaned and validated, and each batch is tested for purity, safety and potency before release.
1.11 Summary
Biotechnology combines “bio”, from biology, with “technology”. It is the use of living systems to make useful goods and provide services, and it treats an organism not as something merely to be studied but as something to be put to work.
The term was coined by Karl Ereky in 1917 and appeared in print in 1919. According to Ereky, biotechnology is a process by which raw material can be biologically upgraded into socially useful products, and he is regarded by one group of scientists as the Father of Biotechnology.
The European Federation of Biotechnology (EFB) defines biotechnology as the integration of natural science and organisms, cells, parts thereof, and molecular analogues, for products and services.
The Organization for Economic Co-operation and Development (OECD) defines biotechnology as the application of science and technology to living organisms, and to their parts, products and models, to alter living or non-living materials for the production of knowledge, goods and services. This is the broadest of the three definitions, since it admits non-living material and counts knowledge as an outcome.
Biotechnology is interdisciplinary. It draws on biochemistry, microbiology, genetics, cell biology and engineering, because no single science can supply the organism, the reactions, the purity and the vessel all at once.
Biotechnology is as old as human civilisation. Wine and beer date from about 6000 BC, and bread and curd from about 4000 BC, made by people who had reliable methods but no knowledge of the organisms responsible.
Domestication of plants and animals, beginning roughly ten thousand years ago, was the earliest deliberate biotechnology. Selective breeding improved disease resistance, taste and yield by choosing which individuals became parents.
Yeasts, moulds and Lactobacillus bacteria were used for centuries to preserve milk, fruits and vegetables. Ancient Indian texts describe somarasa; the Indus Valley and Indo-Aryan civilisations used fermentation for foods, medicines and cleanliness; and the Babylonians, Sumerians and Egyptians prepared alcohol and vinegar, using yeast for wine and beer and bacteria for yoghurt.
Robert Hooke observed and named cells in cork in 1665, and Anton van Leeuwenhoek was the first to see living microorganisms. Without the microscope, the agent of fermentation could never have been identified.
Gregor Mendel, the Father of Genetics, worked on pea plants (Pisum sativum) between 1856 and 1863 and showed that traits are controlled by discrete factors. He established the Law of Dominance, the Law of Segregation and the Law of Independent Assortment, laying the foundation of classical genetics.
Alexander Fleming discovered penicillin in 1928 from the mould Penicillium notatum, while working with Staphylococcus. This began the antibiotic era and won him a share of the Nobel Prize in Physiology or Medicine in 1945.
Rosalind Franklin and Maurice Wilkins used X-ray diffraction to show that DNA is helical, and their evidence enabled James Watson and Francis Crick to propose the double helix model in 1953. DNA has two antiparallel polynucleotide strands held together by specific base pairing, adenine with thymine by two hydrogen bonds and guanine with cytosine by three, which accounts for Chargaff’s equivalence rule. Watson, Crick and Wilkins received the Nobel Prize in 1962.
Werner Arber and Hamilton O. Smith discovered and characterised restriction enzymes, which cut DNA at specific recognition sequences called restriction sites and are therefore called molecular scissors. Herbert Boyer and Stanley Cohen made the first recombinant DNA molecules in 1973, using restriction enzymes to cut DNA, plasmids as vectors, DNA ligase to join fragments, and transformation to introduce them into Escherichia coli.
Louis Pasteur is the Father of Modern Biotechnology. In 1857 he showed experimentally that living organisms take part in all fermentative processes and that each fermentation is associated with a specific organism, giving rise to the germ theory of fermentation.
Pasteur found that fermentation is arrested when air is passed through the fermenting fluid, and he introduced the terms aerobic and anaerobic. In 1861 he showed that yeast consumes far more glucose anaerobically than aerobically, because the yield falls from about 38 ATP to about 2 ATP per glucose. This is the Pasteur Effect.
Industrially, the Pasteur Effect is used in two stages: yeast is first grown in an aerated broth to build up biomass, and air is then excluded so that the sugar is converted to alcohol rather than to more yeast.
In 1863 Pasteur prevented wine spoilage by heating the wine to 60 °C and cooling it immediately. This process, pasteurization, kills most spoilage and disease-causing organisms without seriously altering the product, and is now applied to many foods and beverages, particularly milk. Rapid cooling is essential because the product is not sterilised and the survivors must not be allowed to multiply.
Traditional biotechnology uses whole organisms and their natural processes, without altering genetic material. It is empirical, slow and limited to what the organism can already do; its products include fermented foods, wine and beer, improved crop varieties, compost and treated sewage.
Modern biotechnology works at the level of the gene, cell and molecule. It is specific, can cross species barriers, and is rapid; its techniques include recombinant DNA technology, cell and tissue culture, gene analysis and hybridoma technology, and its products include Bt cotton, Golden Rice, the recombinant hepatitis-B vaccine, bioremediation and micropropagation.
The kitchen is the first biotechnology laboratory, because it supplies all four elements of a biotechnological process — a chosen biological agent, a substrate, controlled conditions and a defined product — without instruments or explanation.
Curd is made by adding a starter culture containing lactic acid bacteria, mainly Lactobacillus, to warm milk. These bacteria convert lactose into lactic acid, the pH falls, and the milk protein casein coagulates to give a thick, semi-solid product.
Curd formation improves the digestibility, taste, texture and nutritional value of milk, increases its vitamin B content, and inhibits harmful microorganisms because the environment is acidic. Milk is boiled and cooled to about 37 °C before the starter is added, so that competitors are removed and the bacteria work at their optimum temperature.
Many Indian foods are kitchen fermentations: idli and dosa from rice and black gram, dhokla and khaman from Bengal gram, bhatura and naan from wheat leavened by yeast, srikhand and mishti dahi from milk, and gundruk, sinki and mesu from vegetables preserved by acidification and drying.
Fermentation is the anaerobic breakdown of an organic substrate, usually a sugar, in which little energy is released and organic end products remain. Industry values it because those incomplete end products are the wanted product, and because no oxygen supply is needed.
Homofermentation yields a single main end product, lactic acid, with no carbon dioxide, and gives 2 ATP per glucose: \(\text{C}_6\text{H}_{12}\text{O}_6 \longrightarrow 2\,\text{C}_3\text{H}_6\text{O}_3 + 2\,\text{ATP}\). It proceeds by glycolysis followed by the reduction of pyruvate to lactic acid by lactate dehydrogenase, a step that regenerates NAD⁺ and keeps glycolysis running.
Lactic acid fermentation is central to the dairy industry, giving curd, yoghurt, cheese and buttermilk, and to the probiotic industry, where the live bacteria are themselves the product and must survive to reach the consumer’s gut.
Heterofermentation yields more than one end product — lactic acid, ethanol and carbon dioxide — proceeds by the pentose phosphate pathway, and releases only 1 ATP per glucose: \(\text{C}_6\text{H}_{12}\text{O}_6 \longrightarrow \text{C}_2\text{H}_5\text{OH} + \text{C}_3\text{H}_6\text{O}_3 + \text{CO}_2 + \text{ATP}\). It matters to the bakery, brewing and alcohol industries.
Alcoholic fermentation converts simple sugars into ethanol and carbon dioxide, mainly by Saccharomyces cerevisiae: \(\text{C}_6\text{H}_{12}\text{O}_6 \longrightarrow 2\,\text{C}_2\text{H}_5\text{OH} + 2\,\text{CO}_2 + 2\,\text{ATP}\). It supports the bioethanol industry, where starch-rich crops must first be hydrolysed to fermentable sugars, and the beverage industry.
Beer is an undistilled alcoholic beverage of 4–8 per cent alcohol, produced from barley or other cereals using Saccharomyces cerevisiae, with starch-rich adjuncts such as wheat and maize.
In malting, barley is cleaned, soaked for two days and germinated for four to six days, so that amylase and protease are formed. The temperature is then raised to about 80 °C to stop germination while leaving the enzymes active, and the seeds are powdered to give malt.
In mashing, powdered malt is mixed with hot water at 55–65 °C. Amylase degrades starch to maltose, dextrin and glucose, and proteases degrade proteins to peptones, peptides and amino acids, giving the fermentation medium called beer wort. Dried female flowers of the hop plant are added for flavour, aroma and a stabilising effect.
Fermentation of the wort is carried out with Saccharomyces cerevisiae in open or closed fermentors at 20–28 °C for 5 to 10 days, converting sugars anaerobically into ethanol and carbon dioxide.
Maturation is carried out in storage tanks at 0–3 °C for roughly a few weeks, during which yeast cells sediment and nitrogenous substances, resins and phosphates precipitate. Chill-proofing then removes residual proteins, which would otherwise leave the beer turbid.
Carbonation is carried out by injecting carbon dioxide, after which the beer is bottled or canned and pasteurized at 60 °C for about 20 minutes.
Good Laboratory Practices (GLP) are the rules governing research, routine and trial experiments in laboratories. They ensure that data are accurate, reliable, reproducible and ethically obtained, and that personnel, the public and the environment remain safe.
GLP rests on four principal components: Management, Quality Assurance including the Animal Ethics and Bioethics Committees, the Study Director, and the National Compliance Monitoring Authority (NCMA). Guidelines for GLP are provided by the Organisation for Economic Co-operation and Development (OECD).
Basic laboratory practices under GLP include prohibiting food and drink, wearing laboratory coats and protective equipment, proper training on instruments, cleanliness and drainage, properly washed glassware, and safe disposal of chemical, biological and radioactive waste.
Quality control tests raw materials, intermediates and finished products against defined specifications. For a biological product this means checking identity, purity, potency, safety and stability, using sampling and full documentation so that a fault can be traced back to its origin.
Good Manufacturing Practices (GMP) are the guidelines followed during manufacture, processing, packaging and storage, so that every batch is consistently produced with the required quality, safety and efficacy. They are essential in industries producing vaccines, antibiotics, enzymes, hormones and recombinant products.
The principles of GMP cover hygienic clean-room facilities, trained personnel with defined responsibilities, standard operating procedures for every step, regular quality control and quality assurance checks, documentation at every stage, and safe storage, packaging and distribution.
GLP and GMP are complementary. GLP makes the evidence behind a product trustworthy; GMP makes the batch that reaches the consumer match the one that was tested.
Flowchart
FC 1.10 – The argument of the chapter in one line: ancient practice without explanation (fermented foods, selective breeding) → the organism is seen (Hooke, Leeuwenhoek) → inheritance is explained (Mendel) → the organism is identified as the agent of fermentation (Pasteur; germ theory, Pasteur Effect, pasteurization) → conditions are controlled in the kitchen and then in the fermenter (curd, beer) → the genetic material itself is manipulated (modern biotechnology) → the process and the product are regulated (GLP, quality control, GMP)
1.12 Key Terms
Biotechnology the use of living systems, their cells, parts or molecular analogues to alter materials and produce useful goods, services and knowledge.
EFB European Federation of Biotechnology, the body that defines biotechnology as the integration of natural science and organisms, cells, parts thereof, and molecular analogues, for products and services.
OECD Organization for Economic Co-operation and Development, the body that defines biotechnology as the application of science and technology to living organisms and their parts, products and models, and that also issues the guidelines for GLP.
Interdisciplinary drawing on more than one branch of science, as biotechnology draws on biochemistry, microbiology, genetics and engineering together.
Domestication the bringing of wild plants and animals under human care and cultivation, the earliest form of deliberate biotechnology.
Selective breeding the improvement of a population by choosing which individuals become parents, so that desired inherited traits accumulate over generations.
Cell the basic unit of which living material is built, first observed and named by Robert Hooke in cork.
Factors Mendel’s name for the discrete inherited units, later called genes, that control traits and pass unchanged from parents to offspring.
Law of Dominance the principle that when two contrasting factors are present, one may mask the expression of the other.
Law of Segregation the principle that the two factors of a pair separate during gamete formation, so that each gamete receives only one of them.
Law of Independent Assortment the principle that different pairs of factors are distributed to gametes independently of one another.
Antibiotic a substance produced by a microorganism that kills or suppresses the growth of other microorganisms.
Penicillin the first antibiotic, discovered by Alexander Fleming from the mould Penicillium notatum.
X-ray diffraction a technique in which the scattering pattern of X-rays passed through a sample is used to work out the arrangement of its atoms, used by Franklin and Wilkins to study DNA.
Double helix the structure of DNA proposed by Watson and Crick, in which two antiparallel polynucleotide strands are coiled around each other.
Antiparallel describing the two strands of DNA, which run in opposite directions to each other.
Chargaff’s equivalence rule the observation that in any sample of DNA the total number of purine bases equals the total number of pyrimidine bases.
Restriction enzyme an enzyme that recognises a specific short sequence in DNA and cuts the molecule at that point; also called a restriction endonuclease.
Restriction site the specific recognition sequence in DNA at which a restriction enzyme cuts.
Molecular scissors the common name for restriction enzymes, reflecting their ability to cut DNA at defined positions.
Restriction–modification system the bacterial defence system, proposed by Arber, in which foreign DNA is cut by restriction enzymes while the cell’s own DNA is protected by methylation.
Methylation the addition of methyl groups to DNA, which in bacteria protects their own recognition sites from being cut.
Recombinant DNA technology the set of techniques by which DNA fragments from different sources are cut, joined and replicated inside a living host cell.
Plasmid a small circular DNA molecule that a bacterium carries in addition to its main chromosome, widely used as a vector.
Vector a DNA molecule used to carry a foreign DNA fragment into a host cell.
DNA ligase the enzyme that joins DNA fragments together to form a continuous molecule.
Transformation the process by which a bacterium takes up DNA from its surroundings and maintains it.
Germ theory of fermentation Pasteur’s principle that fermentation is brought about by living microorganisms, each type of fermentation being carried out by a specific organism.
Aerobic describing an organism or process that requires the presence of oxygen.
Anaerobic describing an organism or process that operates in the absence of oxygen.
Pasteur Effect the suppression of fermentation, and the fall in the rate of glucose consumption, when oxygen is supplied to a fermenting culture such as yeast.
ATP adenosine triphosphate, the molecule in which a cell stores and transfers the energy released by respiration.
Pasteurization a controlled heat treatment in which a food or beverage is heated to a moderate temperature for a defined time and cooled rapidly, destroying most spoilage and disease-causing organisms without seriously altering the product.
Traditional biotechnology the use of whole living organisms and their natural processes to obtain useful products, without any deliberate alteration of genetic material.
Modern biotechnology the use of techniques acting directly on genetic material, cells and molecules to obtain products that natural organisms could not provide on their own.
Tissue culture the growth of cells, tissues or whole organisms under sterile conditions in a defined nutrient medium.
Bioremediation the use of selected microorganisms to break down pollutants such as oil spills and industrial effluents.
Micropropagation the raising of large numbers of identical, disease-free plants from a small piece of tissue.
Starter culture a small quantity of a previously fermented product, or a prepared culture, added to a fresh substrate to begin fermentation with the desired organism.
Lactic acid bacteria (LAB) bacteria, mainly species of Lactobacillus, that ferment sugars to lactic acid and are responsible for setting curd.
Lactose the sugar naturally present in milk, which lactic acid bacteria ferment to lactic acid.
Casein the milk protein that coagulates when the pH falls, converting liquid milk into set curd.
Whey the watery liquid that separates from the curd mass on standing.
Fermentation the anaerobic breakdown of an organic substrate, usually a sugar, in which a small amount of energy is released and organic compounds remain as end products.
Homofermentation fermentation in which glucose is broken down to a single main end product, lactic acid, with no carbon dioxide and a yield of 2 ATP.
Glycolysis the pathway in which glucose is converted to two molecules of pyruvate, yielding 2 ATP and 2 NADH.
Lactate dehydrogenase the enzyme that reduces pyruvate to lactic acid, regenerating NAD⁺ so that glycolysis can continue.
Lactic acid fermentation the anaerobic conversion of a sugar, typically lactose, into lactic acid by lactic acid bacteria.
Probiotic a food or supplement containing live beneficial microorganisms that promote gut health.
Heterofermentation fermentation in which glucose is converted, by the pentose phosphate pathway, into more than one end product — lactic acid, ethanol and carbon dioxide — with a yield of 1 ATP.
Alcoholic fermentation the anaerobic conversion of simple sugars into ethanol and carbon dioxide by yeast, with the release of a small amount of energy.
Bioethanol ethanol produced by the fermentation of plant-derived sugars and used as a renewable fuel or fuel additive.
Beer an undistilled alcoholic beverage of 4–8 per cent alcohol content, produced by fermenting barley or other cereals with Saccharomyces cerevisiae.
Adjuncts starch-rich materials such as wheat and maize added to the brewing process to enhance fermentation.
Malting the controlled soaking and germination of barley, arrested by heating to about 80 °C, which yields grain rich in active amylase and protease.
Malt the powdered, enzyme-rich product obtained at the end of malting.
Amylase the enzyme formed during germination that hydrolyses starch to maltose, dextrin and glucose.
Protease the enzyme formed during germination that hydrolyses proteins to peptones, peptides and amino acids.
Mashing the mixing of powdered malt with hot water at 55–65 °C, during which the malt enzymes break down starch and protein.
Beer wort the sugar-rich medium obtained at the end of mashing, which serves as the substrate for fermentation.
Hops the dried female flowers of the hop plant, added during brewing to provide flavour, aroma and a stabilising effect.
Maturation the storage of fermented beer at 0–3 °C, during which yeast sediments and unwanted substances precipitate.
Chill-proofing the removal of residual proteins from matured beer, which would otherwise make it turbid.
Carbonation the injection of carbon dioxide into matured beer to give a uniform degree of effervescence.
GLP Good Laboratory Practices, the rules governing laboratory work so that data are accurate, reliable, reproducible and ethically obtained, and personnel and the environment stay safe.
Quality Assurance (QA) the internal monitoring system or committee that checks whether GLP procedures are being followed in day-to-day laboratory work.
Study Director the person responsible for the overall conduct of a research or safety study, for correct recording of data and for adherence to safety rules.
NCMA National Compliance Monitoring Authority, the national regulatory body that approves safety studies on the health and environmental impact of new biotechnology products.
Animal Ethics Committee a quality assurance body that must clear any research protocol involving experiments on animals.
Bioethics Committee a quality assurance body that reviews the ethical acceptability of a proposed study.
Quality control (QC) the testing of raw materials, intermediates and finished products against defined specifications, so that only material meeting the standard is released.
Potency the strength of the biological activity of a product, which for a vaccine or an enzyme matters more than the quantity present.
GMP Good Manufacturing Practices, the guidelines governing manufacture, processing, packaging and storage so that every batch is consistently produced to the required quality, safety and efficacy.
SOP Standard operating procedure, a written approved step-by-step instruction for an operation, followed identically on every occasion.
Clean room a manufacturing area designed and maintained to keep airborne contamination below a defined level.
1.13 Points to Ponder
Three definitions of biotechnology were given in this chapter, framed by an engineer, a scientific federation and an economic organisation. Does the fact that a subject needs three official definitions suggest that its boundaries are genuinely unclear, or only that different users need different emphases?
Ereky spoke of raw material being “biologically upgraded”. If a laboratory one day assembles a functioning cell from chemically synthesised parts, would that still be an upgrading of raw material, and would any of the three definitions in Section 1.1 comfortably cover it?
A cook setting curd controls the organism, the substrate, the temperature and the time, but could not name any of them. Is understanding necessary for technology, or only for improving it?
Pasteur’s conclusion — that a living organism causes fermentation — was resisted by leading chemists of his day. What made the older, purely chemical explanation so persuasive, and how would you have tested between the two views using only nineteenth-century equipment?
Traditional fermented foods differ sharply from region to region, following the crops, the climate and the local microorganisms. What is lost when a regional product is standardised into a single industrial strain and process, and what is gained?
Fermented food recipes have been developed and refined over centuries by communities that kept no written records and hold no patents. If a company isolates the organism from such a food and patents an improved strain, what claim, if any, does the community of origin have?
Alcohol production is one of the world’s largest fermentation industries and also a source of considerable social harm. Should a biotechnology course teach the production of alcoholic beverages in technical detail, and does understanding a process amount to endorsing it?
Pasteurization reduces the microbial load in milk but does not sterilise it, and it slightly alters taste and nutritional content. Some consumers argue for raw milk on those grounds. How would you weigh a small loss in quality for everyone against a small risk of serious infection for a few?
Fleming noticed a contaminated plate that many others might have discarded. How much of a scientific discovery is chance, and what is it in a researcher’s preparation that turns an accident into a finding?
Restriction enzymes evolved as a bacterial defence against viruses and became the central tool of genetic engineering. What does this suggest about where the next generation of biotechnological tools is most likely to be found?
Modern biotechnology can move a gene between unrelated species in a single step, something no amount of breeding could achieve. Is crossing the species barrier a difference of degree from selective breeding, or a difference of kind that deserves separate regulation?
Suppose yeast were engineered to ferment starch directly, making malting unnecessary. Trace what such a change would do to the brewing process, to the cost of beer, and to the barley-growing and malting industries. Who would gain and who would lose?
GLP places the decision on whether an experiment is ethically acceptable outside the hands of the researcher who wants the result. Is this a sensible safeguard against self-interest, or an obstacle that slows down work that could save lives?
Compliance with GLP and GMP adds substantially to the cost of a medicine, and that cost is ultimately paid by the patient. In a country with limited health spending, how would you argue for maintaining the full standard rather than a cheaper local one?
A probiotic product claims to improve gut health. Using the ideas of identity, purity, potency and stability from Section 1.10, what evidence would you insist on before allowing that claim to appear on the label?
The chapter presented biotechnology as a story of increasing control — over the organism, the conditions and finally the genetic material itself. Is there any point at which greater control over living systems stops being an unqualified good?
1.14 Objective Questions
- Karl Ereky’s book in which the term biotechnology first appeared in print was published in:
- 1857
- 1909
- 1919
- 1928
- Which definition of biotechnology explicitly allows the material being altered to be non-living?
- Ereky’s definition
- the EFB definition
- the OECD definition
- none of the three
- The phrase “parts thereof” in the EFB definition makes it possible to include which of the following as biotechnology?
- a detergent containing an isolated bacterial enzyme
- a purely chemical fertiliser
- a mechanical milking machine
- a refrigerated storage unit
- Biotechnology is described as an interdisciplinary subject because it requires:
- only microbiology
- biology together with chemistry, but not engineering
- biology, biochemistry, microbiology and engineering together
- engineering alone at industrial scale
- Selective breeding practised by ancient farmers is classified as biotechnology because it:
- used laboratory equipment
- altered the inherited make-up of a population to obtain a more useful product
- involved transfer of genes between species
- required knowledge of Mendel’s laws
- Mummification practised by the Egyptians preserved the body by:
- encouraging useful microorganisms
- fermenting the tissues
- removing the water that microorganisms require
- lowering the pH of the tissues
- Pasteur introduced the terms aerobic and anaerobic after observing that:
- fermentation was arrested when air was passed through the fermenting fluid
- yeast cells multiplied faster in sealed vessels
- lactic acid was formed only in the presence of oxygen
- heating the wine prevented spoilage
- According to the Pasteur Effect, a yeast culture deprived of oxygen will:
- consume less glucose and release more energy per molecule
- consume more glucose and release less energy per molecule
- consume less glucose and release less energy per molecule
- consume the same glucose but produce more yeast
- In industrial alcohol production, yeast is first grown in an aerated broth in order to:
- increase the alcohol yield directly
- build up a large population of yeast cells
- destroy contaminating bacteria
- raise the temperature of the medium
- Milk is boiled and then cooled to about 37 °C before adding the starter because boiling:
- supplies the lactose needed by the bacteria
- removes competing organisms, while cooling brings the milk to the optimum growth temperature
- coagulates the casein in advance
- lowers the pH of the milk
- The setting of milk into curd is caused directly by:
- the multiplication of bacterial cells in the milk
- the coagulation of casein as the pH falls
- the release of carbon dioxide
- the separation of whey
- Reduction of pyruvate to lactic acid during homofermentation is essential because it:
- yields two additional molecules of ATP
- releases carbon dioxide
- regenerates NAD⁺ so that glycolysis can continue
- lowers the temperature of the medium
- The ATP yield per molecule of glucose in heterofermentation is:
- 1 ATP
- 2 ATP
- 4 ATP
- 38 ATP
- Which of the following is produced in heterofermentation but not in homofermentation?
- lactic acid
- ATP
- carbon dioxide
- NAD⁺
- Starch-rich crops must be treated before alcoholic fermentation can begin, because yeast cannot:
- survive in the presence of starch
- ferment a polysaccharide directly
- grow below 20 °C
- produce carbon dioxide from glucose
- During malting, the temperature is raised to about 80 °C in order to:
- destroy the amylase and protease formed
- stop germination while leaving the enzymes active
- convert starch into maltose
- sterilise the beer wort
- The mashing temperature of 55–65 °C is chosen because it is the range in which:
- yeast ferments most rapidly
- hops release the maximum aroma
- the malt enzymes act rapidly without being denatured
- proteins precipitate out of the wort
- The products of protein breakdown obtained at the end of mashing are:
- maltose, dextrin and glucose
- peptones, peptides and amino acids
- ethanol and carbon dioxide
- resins and phosphates
- Chill-proofing is carried out during beer production in order to remove:
- residual proteins that would make the beer turbid
- surviving yeast cells
- excess carbon dioxide
- bitter compounds contributed by hops
- Carbon dioxide is injected into matured beer chiefly to:
- kill spoilage organisms
- restart fermentation in the bottle
- give every bottle the same measured degree of effervescence
- lower the pH of the beer
- Guidelines for GLP are provided by:
- EFB
- OECD
- NCMA
- the Study Director
- Which of the following is the only component of GLP that operates from outside the organisation?
- Management
- Quality Assurance
- the Study Director
- the National Compliance Monitoring Authority
- The Animal Ethics Committee and the Bioethics Committee are examples of:
- quality assurance bodies
- national regulatory authorities
- manufacturing units
- standard operating procedures
- A written, approved, step-by-step instruction followed identically on every occasion is called:
- a batch record
- a standard operating procedure
- a specification
- a compliance certificate
- For a vaccine, the quality control parameter that measures the strength of biological activity rather than the quantity present is:
- identity
- purity
- potency
- stability
- Which pair correctly matches the system with its unit of attention?
- GLP — the batch; GMP — the study
- GLP — the study; GMP — the batch
- GLP — the product; GMP — the data
- GLP — the batch; GMP — the specification
- For the following questions, choose from the following options:
- Both Assertion and Reason are true, and Reason is the correct explanation of Assertion.
- Both Assertion and Reason are true, but Reason is not the correct explanation of Assertion.
- Assertion is true, but Reason is false.
- Both Assertion and Reason are false.
- Assertion (A): The kitchen can reasonably be called the first biotechnology laboratory. Reason (R): A kitchen supplies a biological agent, a substrate, controlled conditions and a defined product.
- Assertion (A): Paneer is not classified as a fermented milk product. Reason (R): In paneer the casein is coagulated by directly adding an acid, without the involvement of any microorganism.
- Assertion (A): Curd keeps longer than the milk from which it was made. Reason (R): The acidic environment produced by lactic acid inhibits the growth of harmful microorganisms.
- Assertion (A): Pasteurized milk must still be kept refrigerated. Reason (R): Pasteurization destroys every microorganism present in the milk.
- Assertion (A): Homofermentation releases more ATP per glucose molecule than heterofermentation. Reason (R): Homofermentation proceeds by glycolysis, whereas heterofermentation proceeds by the pentose phosphate pathway.
- Assertion (A): Hops are described as a stabilising agent as well as a flavouring. Reason (R): The compounds that give hops their bitterness also discourage the growth of many spoilage bacteria.
- Assertion (A): Beer wort is prepared before the yeast is added, and not after. Reason (R): Yeast cannot ferment the starch of barley grain directly, so the starch must first be hydrolysed to sugars.
- Assertion (A): Traditional biotechnology has been made obsolete by modern biotechnology. Reason (R): Modern techniques can transfer a chosen gene between unrelated species in a single step.
- Assertion (A): GLP requires observations to be recorded at the time they are made. Reason (R): A record made later cannot prove what was actually observed, and copying introduces the chance of transcription errors.
- Assertion (A): Quality control alone is sufficient to guarantee that every batch of a biotechnological product is safe. Reason (R): Quality control tests a sample of the product against a specification after it has been made.
- The term Biotechnology was coined by Karl Ereky in the year:
- 1901
- 1917
- 1928
- 1945
- Which organisation defines biotechnology as the integration of natural sciences and organisms for products and services?
- WHO
- UNESCO
- EFB
- FAO
- According to Karl Ereky, biotechnology primarily involves:
- genetic modification of plants
- industrial use of microbes
- biological upgrading of raw materials into useful products
- application of enzymes in medicine
- Robert Hooke first observed cells in:
- onion peel
- animal tissue
- cork
- bacterial culture
- Living cells were first observed by:
- Robert Hooke
- Louis Pasteur
- Anton van Leeuwenhoek
- Gregor Mendel
- Gregor Mendel’s experiments were performed on:
- wheat plants
- pea plants
- maize plants
- rice plants
- Penicillin was discovered from the mould:
- Aspergillus
- Rhizopus
- Penicillium notatum
- Saccharomyces
- Pasteurization involves heating food to approximately:
- 100 °C
- 80 °C
- 60 °C
- 40 °C
- Recombinant DNA technology was first demonstrated by:
- Watson and Crick
- Arber and Smith
- Boyer and Cohen
- Pasteur and Fleming
- Alcoholic fermentation is primarily carried out by:
- Lactobacillus
- Rhizobium
- Saccharomyces cerevisiae
- Escherichia coli
- The discovery of penicillin marked the beginning of:
- the vaccination era
- the biotechnology era
- the antibiotic era
- the genetic engineering era
- Louis Pasteur is regarded as the Father of Modern Biotechnology because he:
- discovered DNA
- proposed the germ theory of fermentation
- discovered restriction enzymes
- developed recombinant DNA technology
- The Pasteur Effect refers to:
- increased ATP production under anaerobic conditions
- arrest of fermentation by oxygen
- increased glucose consumption in the absence of oxygen
- conversion of sugar into ethanol
- Restriction enzymes are also called molecular scissors because they:
- replicate DNA
- ligate DNA fragments
- cut DNA at specific sites
- protect DNA from mutation
- The kitchen is considered the first biotechnology laboratory because of:
- genetic engineering
- enzyme purification
- use of microbes in food fermentation
- industrial processing
- Homofermentation produces mainly:
- ethanol and \(\text{CO}_2\)
- lactic acid only
- ethanol only
- lactic acid and ethanol
- Heterofermentation results in the production of:
- lactic acid only
- ethanol only
- lactic acid, ethanol and \(\text{CO}_2\)
- \(\text{CO}_2\) only
- Which step in beer production stops germination but preserves the enzymes?
- mashing
- fermentation
- malting
- carbonation
- GLP mainly ensures:
- increased production
- profitability
- safety and reliability of experimental data
- faster experiments
- Curd formation from milk is an example of:
- aerobic respiration
- alcoholic fermentation
- lactic acid fermentation
- photosynthesis
- GMP is MOST essential during:
- laboratory teaching
- school experiments
- industrial production of vaccines
- field trials
- For the following questions, choose from the following options:
- Both Assertion and Reason are true, and Reason is the correct explanation of Assertion.
- Both Assertion and Reason are true, but Reason is not the correct explanation of Assertion.
- Assertion is true, but Reason is false.
- Both Assertion and Reason are false.
- Assertion (A): Fermentation is considered a biological process rather than a purely chemical reaction. Reason (R): Living microorganisms such as yeast actively carry out fermentation.
- Assertion (A): Pasteurization is an important process in food preservation. Reason (R): It involves controlled heating to reduce the number of harmful microorganisms.
- Assertion (A): Modern biotechnology differs fundamentally from traditional biotechnology. Reason (R): Modern biotechnology relies on recombinant DNA technology and genetic engineering techniques.
- Assertion (A): Beer and wine production depends on anaerobic fermentation. Reason (R): In the absence of oxygen, yeast converts sugars into ethanol and carbon dioxide.
- Assertion (A): Louis Pasteur disproved the idea that fermentation occurs spontaneously. Reason (R): He demonstrated that microorganisms are responsible for converting sugars into alcohol or acids.
- Assertion (A): Industrial fermentation is commonly used to produce products such as alcohol, organic acids and enzymes. Reason (R): Microorganisms metabolise sugars under controlled conditions during fermentation.
- Assertion (A): Yeast is widely used in bread making to improve texture and volume. Reason (R): Carbon dioxide released during yeast fermentation causes the dough to rise.
- Assertion (A): Following Good Laboratory Practices (GLP) is essential in research laboratories. Reason (R): GLP ensures accuracy, reliability and proper documentation of experimental data.
- Assertion (A): Traditional biotechnology includes advanced techniques such as gene editing and tissue culture. Reason (R): Traditional biotechnology primarily involves age-old fermentation practices using whole organisms.
- Assertion (A): Fermentation is preferred in certain industries for large-scale production of useful products. Reason (R): Fermentation always involves aerobic breakdown of glucose by microorganisms.
- Assertion (A): Good Manufacturing Practices (GMP) play a crucial role in ensuring product quality. Reason (R): GMP focuses only on laboratory research and does not apply to industrial production.
- Assertion (A): Compliance with GMP is essential for the approval of biotechnological products. Reason (R): GMP ensures consistency, hygiene, proper documentation and quality control during manufacturing.
Fill in the blank: The microorganism commonly used in curd formation is __________.
Fill in the blank: Alcoholic fermentation is mainly carried out by the fungus biologically called __________.
Fill in the blank: The term biotechnology was coined by __________.
Fill in the blank: The mould used for penicillin production is __________.
Fill in the blank: Barley is mainly used as the __________ grain in beer production.
Fill in the blank: Restriction enzymes are also known as __________ scissors.
Fill in the blank: The enzyme that joins DNA fragments is called __________.
Fill in the blank: The scientist known as the Father of Modern Biotechnology is __________.
Fill in the blank: The absence of oxygen is essential for __________ fermentation.
Fill in the blank: The conversion of milk into curd is an example of __________ acid fermentation.
Fill in the blank: Pasteurization involves heating milk to about __________ °C for a short time.
Fill in the blank: Homofermentation results primarily in the formation of __________.
Fill in the blank: Heterofermentation produces lactic acid, ethanol and __________.
Fill in the blank: The anaerobic breakdown of glucose into ethanol and \(\text{CO}_2\) is called __________ fermentation.
Fill in the blank: Beer production uses the dried female flowers of the __________ plant.
Fill in the blank: The first organism used in recombinant DNA experiments was __________ coli.
Fill in the blank: Barley germination is stopped during __________ to preserve the enzymes.
Fill in the blanks: Good Laboratory Practices ensure __________ and __________ of experimental data.
Fill in the blank: Good Manufacturing Practices mainly apply to __________ level production.
State whether true or false: Lactobacillus* converts lactose into lactic acid.*
State whether true or false: Yeast produces carbon dioxide during bread making.
State whether true or false: Penicillin was the first antibiotic discovered.
State whether true or false: Saccharomyces cerevisiae* is widely used in biotechnology industries.*
State whether true or false: Pasteurization kills all microorganisms present in milk.
State whether true or false: Fermentation can occur in the presence of oxygen.
State whether true or false: Yeast is widely used in bread making to improve texture and volume.
State whether true or false: GLP guidelines apply only to industrial manufacturing units.
State whether true or false: Homofermentation produces both ethanol and lactic acid.
State whether true or false: GMP ensures product quality and safety during production.
1.15 Check Your Concepts
Ereky’s definition of biotechnology mentions no microorganism and no gene, yet it still covers a modern fermenter producing a recombinant protein. Explain how one sentence written in 1917 manages to include work that did not exist for another fifty years.
A company treats contaminated soil with selected bacteria that break down spilled diesel. Which of the three definitions given in this chapter covers this work most directly, and which phrase in that definition makes the difference?
Why is biotechnology described as interdisciplinary rather than as a branch of biology? Support your answer by naming one contribution each from biochemistry, microbiology and engineering to a single fermentation process.
Ancient producers could neither see nor name the organism they depended on. Explain how, in spite of this, practices such as adding a starter, covering the vessel and keeping it warm amounted to a genuine control of the organism.
Fermentation preserves food by encouraging a microorganism, while mummification and salting preserve by excluding all of them. Compare the two strategies, and give one modern food product that uses each.
What would have been impossible in the development of biotechnology if the microscope had never been invented? Give two specific consequences, drawing on the work of Pasteur.
Mendel’s laws explain what ancient farmers had already been doing for thousands of years. State clearly what the laws added that selective breeding by eye could not provide.
Fleming’s discovery of penicillin is often described as accidental. Identify what in the situation was accidental and what was not, and explain why the same contaminated plate would have taught most people nothing.
The structure of DNA is said to have explained its own function. Explain what is meant by this, using the idea of complementary base pairing.
Restriction enzymes evolved as a bacterial defence against viruses. Explain how a bacterium avoids cutting its own DNA, and why this defence system turned out to be so valuable to genetic engineers.
Arrange in correct historical order and state briefly what each step made possible: (i) the first recombinant DNA molecules, (ii) the naming of the cell, (iii) the germ theory of fermentation, (iv) the double helix model, (v) Mendel’s laws of inheritance.
Leading chemists of Pasteur’s time held that yeast was a by-product of fermentation rather than its cause. Describe an experiment Pasteur could have performed, using only nineteenth-century equipment, that would distinguish between the two views.
A student says that the Pasteur Effect proves yeast prefers oxygen. Rewrite this statement so that it is scientifically accurate, and explain what actually happens to glucose consumption and ATP yield when oxygen is supplied.
Explain why a brewer deliberately runs the same organism under two opposite conditions at two different times in the same process. What is being obtained in each stage?
Why is pasteurization carried out at a moderate temperature rather than at boiling point, when boiling would kill more organisms? Frame your answer as a compromise between two competing requirements.
What would happen to pasteurized milk if it were left standing warm instead of being cooled immediately? Explain the reasoning in terms of the organisms that survive the treatment.
Compare traditional and modern biotechnology under the headings: level of working, species barrier, speed of improvement and regulation required.
Classify each of the following as traditional or modern biotechnology, with a one-line reason: (a) composting kitchen waste, (b) raising virus-free potato plants from shoot tips, (c) producing vinegar from fruit juice, (d) developing a crop variety that carries a bacterial gene for insect resistance.
A kitchen is claimed to satisfy all four requirements of a biotechnological process. Name the four requirements and identify each of them in the making of curd.
Arrange the following in the correct sequence for setting curd from fresh milk, and state what would go wrong if steps two and three were interchanged: add starter culture; boil the milk; leave undisturbed in a warm place; cool to about 37 °C.
Trace the chain of cause and effect from lactose to set curd, naming the organism, the product formed, the change in pH, and the protein affected.
Why does curd set readily in summer but often fail to set in winter, even when the same starter is used? What single change would correct this?
Paneer and curd both end with coagulated casein, yet only one of them is a fermentation. Explain the difference, and state what would happen to the taste and keeping quality of each.
Study Fig. 1.5 in this chapter. Label the two phases formed when set curd is left standing, and explain what each phase contains and why they separate.
Explain why the reduction of pyruvate to lactic acid is essential for a lactic acid bacterium, even though this step yields no ATP of its own.
Lactic acid is a waste product for the bacterium that makes it. Give three distinct ways in which this waste product is useful to the food industry.
Compare homofermentation and heterofermentation with respect to end products, gas released, pathway followed and ATP yield. Then state which of the two a baker would prefer, and why.
In a probiotic product the organism is the product itself, whereas in beer the yeast is merely a processing agent. Explain how this difference changes the manufacturing requirements for the two products.
Match the following, writing the correct pairs: | Column A | Column B | |—|—| | a. Amylase | i. starch-rich materials added to enhance fermentation | | b. Hops | ii. removal of residual proteins from matured beer | | c. Chill-proofing | iii. hydrolyses starch to maltose, dextrin and glucose | | d. Adjuncts | iv. medium obtained at the end of mashing | | e. Beer wort | v. provides flavour, aroma and a stabilising effect |
Explain why barley is germinated at all in beer production, and why that germination must then be stopped rather than allowed to continue.
A brewery’s kiln overheats during malting and the grain reaches 105 °C. Predict what will be observed at the mashing stage and at the fermentation stage, and explain the connection between the fault and each observation.
Arrange the five steps of beer production in order, and for each state the one condition that must be controlled most carefully and what would go wrong if it drifted.
Draw and label a closed fermentor used for beer production, marking the wort inlet, the cooling jacket, the carbon dioxide outlet, the sampling valve and the point at which yeast collects.
Fermentation itself produces carbon dioxide, yet beer is carbonated artificially before packing. Explain this apparent contradiction.
A student writes that GLP exists to make experiments succeed. Correct this statement, and explain what GLP is actually designed to guarantee about an experiment’s results.
Explain why GLP relies on both an internal monitoring body and an external regulatory authority, rather than on either one alone.
Take any three of the seven basic laboratory practices under GLP and state, for each, whether it protects the worker, the experiment, or the world outside the laboratory.
Distinguish between quality assurance and quality control, using a single manufacturing example in which both are applied.
Why is it insufficient to test only the finished batch of a biotechnological product? Name three earlier points at which testing should occur and state what is checked at each.
A vaccine batch passes the tests for identity and purity but fails the test for potency. Explain what this result means and why the batch cannot be released.
Explain why GMP places such emphasis on standard operating procedures, using the idea that a process must not change when the operator changes.
GLP and GMP are described as complementary rather than as alternatives. Explain what each would fail to guarantee if the other were absent.
A family has been making curd at home. Which microorganism is responsible?
Ancient Egyptians used fermentation to produce beer. Which microorganism is involved?
What is the primary grain used in beer production?
What is Louis Pasteur famously known for in microbiology?
Which major discovery of 1953 advanced modern biotechnology?
Who laid the foundation of recombinant DNA technology?
Name two products commonly produced through fermentation.
Explain the role of Lactobacillus* in dairy fermentation.*
Why are the dried female flowers of the hop plant used in beer?
How is Saccharomyces cerevisiae* used in biotechnology?*
Why is milk pasteurized before packaging?
Why is it important to follow GLP in research laboratories?
A batch of beer has a sour taste. What could be the cause, and how could it be prevented?
What factors must be considered while scaling up yoghurt production?
Compare the roles of yeast and bacteria in fermentation.
What is the main goal of GMP in biotechnology industries?
Why are microorganisms important in food biotechnology?
Interpret the significance of pasteurization in today’s food industry.
How have ancient fermentation practices influenced modern biotechnology?
What are the key parameters to be monitored during fermentation?
What safety protocols are essential in a biotechnology laboratory?
Why are GLP and GMP crucial for the approval of biotechnological products?
Outline the process of developing a new fermented food product.
Design a fermentation process for a novel probiotic beverage.
Plan a sustainable model for a biotechnology-based food production unit.
During a classroom discussion, a teacher states that biotechnology is not a modern discovery but has roots in ancient human practices. Students are asked to think about daily life activities that unknowingly involve biological processes. Using suitable examples, explain how biotechnology existed long before the development of modern laboratories and scientific tools.
Maya, a biotechnology student, argues that fermentation should be considered a chemical reaction because it involves the conversion of sugars into alcohol or acids. Another student, Sudipta, disagrees and says it is a biological process. Based on your understanding, explain why fermentation is classified as a biological process and not merely a chemical reaction.
A rural entrepreneur plans to set up a small biofuel unit using sugarcane waste — bagasse and molasses — as raw material. He wants to use fermentation technology to produce ethanol as an alternative fuel. Explain how fermentation can be applied in this process, and what conditions must be maintained for efficient ethanol production.
A family attempts to prepare curd during winter by adding a small amount of starter culture to lukewarm milk, but the milk fails to set even after several hours. Considering the environmental conditions and microbial activity, suggest practical steps that can be taken to ensure proper curd formation.
During a laboratory experiment involving microbial culture, contamination was observed in multiple samples, leading to inaccurate results. The laboratory supervisor emphasised strict adherence to Good Laboratory Practices (GLP). Explain how GLP guidelines can help prevent contamination and ensure reliable experimental outcomes.
During beer production, improper temperature control during the fermentation and maturation stages resulted in poor flavour, turbidity and reduced shelf life of the final product. Analyse which stages of production were affected, and explain the consequences of these errors.
A start-up company wants to produce beer on a commercial scale using locally available grains. Design a detailed process flow including malting, mashing, fermentation and maturation, highlighting the importance of each step.
1.16 Case-Based Questions
- Archaeologists excavating a settlement site recover a large storage jar whose inner surface carries a dried residue. Chemical analysis of the residue shows the breakdown products of cereal starch together with traces of an alcohol. Nothing at the site suggests any knowledge of microorganisms, and no writing survives to describe how the jar was used. A newspaper reports the finding under the headline “Ancient people practised biotechnology”.
- Is the headline justified? Answer using any one of the definitions of biotechnology given in this chapter, and quote the phrase in it that supports your answer.
- The residue contains breakdown products of starch as well as alcohol. Explain why both would be expected, given that yeast cannot ferment starch directly.
- Name the organism most likely responsible for the alcohol, and state the conditions inside the sealed jar that would have favoured it.
- State one thing the people who used this jar could do, and one thing they could not do, in comparison with a modern brewer.
- A class carries out the ISC practical on the effect of temperature on the curdling of milk. Three flasks of boiled milk are cooled and inoculated with the same quantity of the same starter culture, then kept at 10 °C, 37 °C and 60 °C respectively for six hours. The flask at 37 °C sets firmly. The flask at 10 °C is still liquid but sets slowly when later moved to a warm room. The flask at 60 °C remains liquid and does not set even after being moved to a warm room overnight.
- Name the organism in the starter culture and the acid it produces.
- Explain why the flask kept at 10 °C eventually set, while the one kept at 60 °C never did.
- State the biochemical change that converts liquid milk into a set gel, naming the protein involved.
- A student proposes adding a fresh spoonful of starter to the 60 °C flask to rescue it. Predict the outcome and justify your prediction.
- To reduce costs, a small brewery decides to replace one third of its malted barley with unmalted maize, which is cheaper and rich in starch. The malting, mashing, fermentation and maturation schedules are left unchanged. The finished beer is found to be low in alcohol, and a large quantity of unfermented material remains in the vessel at the end of fermentation. The yeast supplier confirms that the yeast culture supplied was healthy and viable.
- Explain why the alcohol content fell, even though the total starch entering the process was unchanged.
- Identify the stage at which the process actually failed, and justify your choice.
- Suggest one change to the process, other than reverting entirely to malted barley, that would allow the maize to be used successfully.
- State why the yeast supplier’s confirmation, though accurate, does not resolve the problem.
- A start-up applies for a government grant reserved for biotechnology enterprises. It has three product lines. The first is an enzyme-based stain remover containing a purified bacterial enzyme and no living cells. The second is a soil treatment in which selected bacteria break down pesticide residues in contaminated farmland. The third is a software package that predicts crop yield from weather data. The grant committee must decide which lines qualify.
- Using the EFB definition, decide whether the first product line qualifies, and name the phrase in the definition on which your decision rests.
- Using the OECD definition, decide whether the second product line qualifies, and explain what is unusual about the material being altered.
- Decide whether the third product line qualifies under any of the three definitions in this chapter, and justify your answer.
- Comment on whether the existence of three different definitions makes the committee’s task easier or harder.
- An inspector visits a laboratory that is conducting safety studies on a new microbial product. The work is competent and the results look convincing. However, the inspector finds that observations are noted on loose sheets during the day and entered into the laboratory notebook each evening; that one series of experiments on laboratory animals was begun before the ethics clearance was issued; and that the person who designed the study, performed it, and signed off the final report is the same individual.
- State which principal component of GLP is missing or weak in each of the three findings.
- Explain why recording observations at the time they are made is treated as more than a matter of neatness.
- Explain why GLP does not allow the researcher who wants a favourable result to be the person who judges whether the study is ethically acceptable.
- The laboratory argues that the data are accurate regardless of these lapses. Evaluate this argument.
- A plant manufacturing two different vaccines on the same premises discovers, during a routine review, that a batch has been dispatched with the labels of the other product. The batch itself meets every specification for the product it actually contains. The batch records are complete and the manufacturing steps were correctly performed. There is no written procedure covering how labels are issued, reconciled and destroyed at the end of a run.
- Which objective of GMP has been breached, even though the product itself is of good quality?
- Explain why a labelling error can be as serious as a contamination error in a vaccine plant.
- Name the principle of GMP that has not been implemented here, and describe the corrective action it implies.
- Explain why blaming the operator would be an inadequate response under a GMP system.
- A company markets a probiotic capsule claiming to contain a defined number of live lactic acid bacteria at the time of consumption. Testing at the factory shows the correct count. Independent testing of the same batch, purchased from a shop eight months later, finds the count has fallen far below the level printed on the pack. The retailer has stored the capsules on an open shelf at room temperature. The pack carries no storage instructions.
- Name the quality control parameter that has failed here, and distinguish it from purity.
- Explain why this failure is more serious for a probiotic than the same failure would be for a fermented drink in which the bacteria are only a processing agent.
- State two measures the company should have taken, one at the testing stage and one at the packaging stage.
- Suggest what a quality control programme should test, and when, in order to catch such a problem before the product is released.
- Gundruk and sinki are traditional fermented vegetable products of the Himalayan region, prepared by communities that have refined the method over many generations without written records. A food company isolates the lactic acid bacteria responsible from a village sample, selects an improved strain, and begins manufacturing a standardised product on an industrial scale. The company applies for protection over its improved strain. Villagers point out that neither the food nor the organism originated with the company.
- Explain the biochemical basis of preservation in gundruk and sinki, naming the organisms and the change they bring about.
- Classify the villagers’ method and the company’s method as traditional or modern biotechnology, giving one reason for each.
- State one clear advantage and one clear disadvantage of replacing the village method with a standardised industrial process.
- Present the arguments on both sides of the company’s claim to protection over the improved strain, without deciding the issue.
- A dairy processing company located in a tropical region noticed that its packaged milk was spoiling within a short duration, especially during the summer months. Despite maintaining refrigeration during transport, microbial contamination remained a major issue affecting shelf life and consumer safety. The quality control team suggested introducing pasteurization before packaging as a preventive measure.
- Define pasteurization.
- Explain how it helps in the preservation of milk.
- Why is immediate cooling necessary after heating?
- Does pasteurization sterilize milk completely? Explain.
- A bakery unit producing large quantities of bread observed that the dough was not rising properly, resulting in dense and hard loaves. The workers had followed the usual procedure but still failed to achieve the desired texture and volume. This led to economic losses and customer complaints.
- What role does yeast play in bread making?
- Which gas is responsible for the dough rising?
- Suggest two reasons for the failure of the dough to rise.
- How can the problem be corrected?
- Two biotechnology industries are studied for comparison. Industry A produces curd and yoghurt using lactic acid bacteria (Lactobacillus), while Industry B produces alcoholic beverages using yeast (Saccharomyces cerevisiae). Both industries rely on fermentation but differ in processes, conditions and products.
- Identify the type of fermentation in both industries.
- Compare their end products.
- Why do their oxygen requirements differ?
- Analyse their industrial significance.
- Daniel has been appointed as a biotechnology consultant to design a small-scale production unit for a probiotic yoghurt drink that promotes gut health. The product must be nutritious, safe and commercially viable.
- Which microorganism or microorganisms would he select, and why?
- Outline the step-by-step production process.
- Mention two key parameters to be controlled during fermentation.
- Suggest measures to ensure product quality using GMP principles.
1.17 Practice With Data
A fermenter is charged with 900 g of glucose, which is completely fermented by yeast according to the equation \(\text{C}_6\text{H}_{12}\text{O}_6 \longrightarrow 2\,\text{C}_2\text{H}_5\text{OH} + 2\,\text{CO}_2 + 2\,\text{ATP}\). Take the molar masses as \(\text{C}_6\text{H}_{12}\text{O}_6 = 180\ \text{g mol}^{-1}\), \(\text{C}_2\text{H}_5\text{OH} = 46\ \text{g mol}^{-1}\) and \(\text{CO}_2 = 44\ \text{g mol}^{-1}\). Calculate (a) the number of moles of glucose fermented, (b) the mass of ethanol formed, (c) the mass of carbon dioxide released, and (d) the mass of ethanol formed per gram of glucose consumed.
The carbon dioxide released in the previous question escapes through a gas lock at 273 K and 1 atm, at which the molar volume of a gas is \(22.4\ \text{dm}^3\ \text{mol}^{-1}\). Calculate the volume of gas released, and comment on why an industrial fermenter must be designed with a gas outlet rather than being sealed completely.
A culture of lactic acid bacteria completely ferments 36 g of glucose by homofermentation, according to \(\text{C}_6\text{H}_{12}\text{O}_6 \longrightarrow 2\,\text{C}_3\text{H}_6\text{O}_3 + 2\,\text{ATP}\), where the molar mass of lactic acid is \(90\ \text{g mol}^{-1}\). Calculate (a) the mass of lactic acid formed, (b) the moles of ATP made available to the bacteria, and (c) the mass of lactic acid that would have been formed had the same quantity of glucose been fermented heterofermentatively, in which only one molecule of lactic acid is produced per molecule of glucose.
Two cultures are each supplied with 0.50 mol of glucose. Culture A is heterofermentative and yields 1 ATP per glucose; culture B is homofermentative and yields 2 ATP per glucose. A third culture, C, respires the same quantity of glucose aerobically at 38 ATP per glucose. Calculate the moles of ATP obtained by each culture, and express the yield of A and of B as a percentage of the yield of C.
A brewery prepares 8000 litres of beer wort containing 105 g of fermentable sugar per litre. Fermentation converts 80 per cent of this sugar to ethanol by the alcoholic fermentation equation. Taking the density of ethanol as \(0.79\ \text{g cm}^{-3}\), calculate (a) the total mass of sugar fermented, (b) the mass and then the volume of ethanol produced, and (c) the alcohol content of the beer as a percentage by volume. State whether the result falls within the 4–8 per cent range quoted for beer, and if not, suggest one change to the wort that would bring it within range.
A malting unit starts with 500 kg of cleaned dry barley. After soaking, germination and kilning, 428 kg of malt is recovered. Calculate the percentage loss in mass, and explain, in terms of what the germinating seed is doing, why some loss of dry matter is unavoidable however carefully the process is run.
The pH of milk inoculated with a starter culture and held at 37 °C was recorded as follows.
| Time (hours) | 0 | 1 | 2 | 3 | 4 | 5 | 6 |
|---|---|---|---|---|---|---|---|
| pH | 6.7 | 6.5 | 6.1 | 5.6 | 5.1 | 4.7 | 4.6 |
(a) Plot pH against time. (b) State during which hour the fall in pH was most rapid, and account for it in terms of the growth of the bacteria. (c) Casein begins to coagulate at about pH 4.7. From the data, estimate when the milk would first have appeared set. (d) Explain why the pH curve begins to flatten towards the end of the run.
- Identical samples of boiled and cooled milk were inoculated with the same starter and held at different temperatures. The time taken for the milk to set firmly was recorded.
| Temperature (°C) | 10 | 20 | 30 | 37 | 45 | 60 |
|---|---|---|---|---|---|---|
| Setting time (hours) | did not set in 12 | 11.0 | 6.5 | 4.0 | 7.5 | did not set at all |
(a) Plot setting time against temperature for those samples that set. (b) Identify the optimum temperature from the data. (c) Explain why the sample at 10 °C did not set within 12 hours but would set if left much longer, whereas the sample at 60 °C would never set. (d) State which single row of this table is most relevant to a family trying to make curd in winter.
A milk sample carrying an initial count of \(6.0 \times 10^{6}\) organisms per millilitre was pasteurized, and the count immediately afterwards was \(6.0 \times 10^{2}\) organisms per millilitre. (a) Calculate the percentage of organisms destroyed. (b) Express the reduction as a factor. (c) The surviving organisms then double every 40 minutes if the milk is left at room temperature. Calculate the count after 4 hours. (d) Use your answer to explain, quantitatively, why rapid cooling is part of the pasteurization procedure.
A probiotic capsule is released with a viable count of \(2.0 \times 10^{10}\) bacteria per capsule. The label guarantees at least \(1.0 \times 10^{9}\) live bacteria per capsule at the end of shelf life. Testing of stored samples gives the following counts.
| Months in storage | 0 | 3 | 6 | 9 | 12 |
|---|---|---|---|---|---|
| Viable count per capsule | \(2.0 \times 10^{10}\) | \(9.0 \times 10^{9}\) | \(4.0 \times 10^{9}\) | \(1.8 \times 10^{9}\) | \(8.0 \times 10^{8}\) |
(a) Plot the viable count on a logarithmic scale against time in storage. (b) Estimate the longest shelf life the company could honestly print on the pack. (c) State which quality control parameter this test is measuring. (d) Explain why a count taken only at the time of manufacture would be insufficient evidence for the label claim.
- During fermentation of a beer batch, the sugar concentration of the wort was measured daily.
| Day | 0 | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
|---|---|---|---|---|---|---|---|---|
| Sugar (g per litre) | 105 | 96 | 74 | 52 | 33 | 21 | 17 | 16 |
(a) Plot sugar concentration against time. (b) Calculate the mean rate of sugar consumption, in g per litre per day, over days 1 to 4. (c) Explain the shape of the curve after day 5 in terms of the yeast and its substrate. (d) The brewer’s schedule allows 5 to 10 days for fermentation. Using the data, justify a decision to end this particular fermentation on day 6.
- A quality control laboratory samples 25 vials from each production batch of a recombinant enzyme and tests them for potency. Over one month, 12 batches are tested and a total of 9 vials fail, distributed as follows: batches 1 to 8 contribute no failures, batch 9 contributes 1 failure, batch 10 contributes 6 failures, and batches 11 and 12 contribute 1 failure each. (a) Calculate the overall percentage of vials that failed. (b) Calculate the percentage of vials that failed within batch 10 alone. (c) Explain why the overall figure gives a misleading picture of the month’s production, and what the batch-wise figures reveal instead. (d) State what action the laboratory should take on batch 10, and why testing a sample rather than every vial is nevertheless the standard practice.