Biotechnology

Question 1

Define biotechnology and give 3 examples

Answer 1

1. Biotechnology involves applying biological organisms or enzymes to the synthesis, breakdown or transformation of materials in the service of people in chemical or industrial processes.
2. Production of food e.g. yogurt, cheese, beer
3. DNA manipulation to produce genetically engineered microorganisms synthesising drugs such as insulin and antibiotics
4. Bio-remediation- use of biological systems to remove soil and water pollutants

Question 2

Define microorganism and give two examples

Answer 2

1. A microscopic organism
2. Fungi - yeast
3. Bacteria

Question 3

Describe the reasons why microorganisms are so useful for biotechnological processes.

Answer 3

1. There are no welfare issues to consider- all is needed is optimum conditions for growth
2. Enormous range which are capable of carrying out many different chemical syntheses or degradations that can be used
3. Genetic engineering allows us to artificially manipulate microorganisms to carry out synthesis reactions that they would not naturally do e.g. produce human insulin
4. Microorganisms have short life cycle and rapid growth rate, huge quantities can be produced in short period of time under the right conditions.
5. The nutrient requirements are often very simple and relatively cheap- modify them so they can utilise materials which would otherwise have been wasted.
6. The conditions which most microorganisms need to grow in include a relatively low temperature, a supply of oxygen and food and the removal of waste gas. They provide their own catalysts. Relatively cheap as no need for high temps and pressures

Question 4

Describe the process of brewing production as an example of biotechnology.

Answer 4

Microorganism- Yeast which respires anaerobically to produce ethanol
Steps:
1. Malting- Barley germinates producing enzymes that break starch molecules down to sugars which yeast can use. Seeds are then killed by slow heating, but enzyme activity retained to produce malt.
2. Mashing- The malt is mixed with hot water and enzymes break down starches to produce wort. Hops are added for flavour and antiseptic qualities. The wort is sterilised and cooled
3. Fermentation- Wort is inoculated with yeast. Temperature maintained for optimum anaerobic respiration. Eventually yeast is inhibited by falling pH, build of ethanol and lack of oxygen.
4. Maturation- the beer is conditioned for 4-29 days and cool temperatures
5. Finishing- the beer is filtered, pasteurised and then bottled or canned with the addition of CO2.

Question 5

Describe the process of baking as an example of biotechnology.

Answer 5

Microorganism- Yeast mixed with sugar and water to respire aerobically. CO2 produced makes bread rise
Steps:
1. The active yeast mixture is added to flour and other ingredients. Mixed and left in warm environment to rise
2. Dough is knocked back (excess air removed), kneaded, shaped and left to rise again
3. Cooked in a hot oven- The CO2 bubbles expand, so the bread rises more. Yeast cells are killed during cooking

Question 6

Describe the process of cheese making as an example of biotechnology.

Answer 6

Microorganism- Bacteria feed on lactose in mild, changing the texture and taste and inhibiting the growth of bacteria which make milk go off.
Steps:
1. The milk is pasteurised to kill off most natural bacteria and homogenised (fat droplets evenly distributed throughout the milk).
2. It is mixed with bacterial cultures and sometimes chymosin enzyme and kept until the milk separates into solid curds and liquid whey.
3. For cottage cheese the curds are separated from the whey, packaged and sold
4. For most cheese, the curds are cut and cooked in the why then strained through draining moulds or cheesecloth. The whey is then used as animal feeds.
5. The curds are put into steel or wooden drums and may be pressed. They are left to dry and mature and ripen before eating as the bacteria continue to act for anything from a few weeks to several years

Question 7

Describe the process yogurt production as an example of biotechnology.

Answer 7

Microorganisms- Bacteria often Lactobacillus bulgaricus (forms ethanal) and Streptococcus thermophilus (forms lactic acid). Both produce extracellular polymers that give yogurt its smooth thick texture.
Steps:
1. Skimmed milk powder is added to milk and the mixture is pasteurised, homogenised and cooled to about 47 degrees.
2. The milk is mixed with a 1:1 ratio of Lb and St and incubated for 4-5 hours at 45 degrees
3. At the end of fermentation the yogurt may be put into cartons at a low temperature or mixed with previously sterilised fruit.
4. Thick-set yogurts are mixed and ferment in the pot

Question 8

Describe one example of a microorganism being used to directly make food for human consumption.

Answer 8

1. Fungus- Fusarium venetatum
2. It is a single-celled fungus grown in large fermenters using glucose syrup as a food source
3. The microorganisms are combined with albumen (egg white) and compressed and formed into meat substitutes. Quorn

Question 9

Describe the advantages of using microorganisms to make food for human consumption.

Answer 9

1. Microorganisms reproduce fast and porduce protein faster than animals and plants
2. Microorganisms have a high protein content with little fat
3. Microorganisms can use a wide variety of waste materials including human and animal wast, reducing costs
4. Microorganisms can be genetically modified to produce the protein required
5. Production of microorganisms is not dependent on weather, breeding cycles etc. It takes place constantly and can be increased or decreased to match demand
6. No welfare issues when growing microorganisms
7. Can be made to taste like anything

Question 10

Describe the disadvantages of using microorganisms to make food for human consumption.

Answer 10

1. Some microorganisms can also produce toxins if the conditions are not maintained at the optimum
2. The microorganisms have to be separated from he nutrient broth and processed to make the food
3. Need sterile conditions that are carefully controlled, adding to the cost
4. Often involve GM organisms and many people have concerns about eating GM food
5. The proteins has to be purified to ensure it contains not toxins or contaminants
6. Many people dislike the thought of eating microorganisms frown on waste
7. Has little natural flavour- needs additives

Question 11

Describe how penicillin is produced commercially, including the name of the microorganism and the conditions needed for the microorganism to grow well.

Answer 11

1. Microorganism- Penicillium chryosogenum- likes high oxygen levels and rich medium and is sensitive to pH and temperature
2. Semi-continuous batch process is used
3. First the fungus grows, then it produces penicillin and finally it is extracted from the medium and purified.
4. The process uses relatively small fermenters because it is difficult to maintain high levels of oxygenation in very large bioreactors.
5. The mixture is continuously stirred to keep it oxygenated
6. Rich nutrient medium
7. The growth medium contains a buffer to maintain the pH at around 6.5
8. Bio reactors are maintained at about 25-27 degrees

Question 12

Describe how human insulin can be produced on a large scale using biotechnology.

Answer 12

1. Genetically engineered bacteria which can make human insulin
2. Bacteria are grown in a fermenter and downstream processing results in a constant supply of pure human insulin
3. Before it used to be extracted from animals pancreas after they had been killed for meat but this had problems e.g. erratic as relied on supply of meat, against some faiths, some people were allergic to animal insulin.

Question 13

Define the term “bioremediation”

Answer 13

1. The use of microorganisms to breakdown pollutants and contaminants in the soil or water

Question 14

Describe two different approaches to bioremediation.

Answer 14

1. Using natural organisms-
   a) many microorganisms naturally break down organic material producing CO2 and water.
   b) Soil and water pollutants are often biological e.g human waste.
   c) If these naturally occurring microorganisms are supported they will break down and neutralise many contaminants.
   d) e.g. In oil spill, nutrients can be added to the water to encourage microbial growth and oil can be dispersed into smaller particles to give maximum surface area for microbial action.
2. GM organisms-
   a) Engineer bacteria to breakdown or accumulate contaminants which they would not naturally encounter.
   b) e.g remove mercury contamination from water, as mercury is very toxic and accumulates in food chains.

Question 15

Describe the inoculating broth way of culturing microorganisms in a laboratory.

Answer 15

Inoculating broth

1. Make a suspension of the bacteria to be grown
2. Mix a known volume with sterile nutrient broth in the flask
3. Stopper the flask with cotton wool to prevent contamination from the air.
4. Incubate at a suitable temperature, shaking regularly to aerate the broth providing oxygen for growing bacteria.

Question 16

Describe the inoculating agar way of culturing microorganisms in a laboratory.

Answer 16

1. The wire inoculating loop must be sterilised by holding it in a Bunsen flame until it glows red hot. It must not be allowed to touch any surfaces as it cools to avoid contamination
2. Dip the sterilised loop in the bacterial suspension.
3. Remove the lid of the Petri dish and make a zig-zag streak across the surface of the agar. Avoid the loop digging into the agar by holding it almost horizontal.
4. Replace the lid of the Petri dish. It should be held down with tape but not sealed completely to allow oxygen to get in, preventing the growth of anaerobic bacteria.
5. Incubate at a suitable temperature

Question 17

How is the food provided for the microorganisms when culturing

Answer 17

1. Using nutrient mediums
2. Either in liquid form- broth or in solid form- agar
3. Nutrients are often added to the agar or broth to provide a better medium for microbial growth.

Question 18

Define aseptic techniques

Answer 18

1. Techniques used to culture microorganisms in sterile conditions so they are not contaminated with unwanted microorganisms

Question 19

Define asepsis

Answer 19

The absence of unwanted bacteria, viruses, and other microorganisms.

Question 20

Describe 3 aseptic techniques and measures used at laboratory and starter culture level.

Answer 20

1. Sterilisation- use a flame, burning off alcohol and steam things in autoclaves
2. Airflow- Limited workspace, keep tubes open for shortest time
3. Access to culture- wash hands, don’t talk, work in limited space. keep closed, open side away from handler

Question 21

Define contaminant

Answer 21

Any unwanted microorganism

Question 22

State 6 aseptic techniques and measures used at large-scale culture level.

Answer 22

1. Steam
2. Stainless steel
3. Sterile additions to culture
4. Filters
5. Minimise number of additives to culture
6. Working practices

Question 23

State 5 reasons why contaminants are a problem in industrial fermentation.

Answer 23

1. They may compete with the culture microorganisms for nutrients and space
2. They may reduce the yield of useful products from the culture
3. They may cause spoilage of the product
4. They may produce toxic chemicals
5. They may destroy the culture microorganism and their products

Question 24

Define fermentation

Answer 24

Anaerobic respiration without the involvement of an electron transport chain

Question 25

Define culture

Answer 25

Growing living matter in vitro, for example, microorganisms in specifically prepared nutrient medium

Question 26

Define pure culture

Answer 26

A culture containing a growth of a single kind of organism free from other organisms.

Question 27

Define mixed culture

Answer 27

A mixed culture is one that contains more than one type of organism growing in a sterile medium, such as agar.

Question 28

Draw, label and annotate a graph of the standard growth curve for microorganisms in a closed culture.

Answer 28

1. Time on x-axis and log of numbers of bacteria on y-axis
2. First mini horizantal line a bit above x-axis, then steep incline, then flat line then slower decline to x-axis
3. Lag phase- when bacteria are adapting to their new environment. They are growing and synthesising the enzymes they need and are not yet reproducing at their maximum rate
4. Log/ exponential phase- the rate of bacterial production is close to or at its theoretical maximum
5. Stationary phase- Occurs when the total growth rate is 0 rate of reproduction= rate of death
6. Death stage- when reproduction has almost ceased and the death rate is larger than reproduction rate

Question 29

Suggest how adding nutrients or removing waste products during different phases would affect the shape of the standard growth curve.

Answer 29

Adding nutrients
1. Lag- no effect
2. Exponential- no change to rate but would last longer
3. Stationary- increased population at this stage and would extend duration if decline is due to lack of nutrients
4. Death- May slow decline if nutrients running out is the issue but no effect if waste is the problem
Removing waste
1. Lag- No effect
2. Exponential- No change to rate but would last longer
3. Stationary- increased population at this stage and would extend duration if decline is due to build up of waste
4. Death- Prevents start of the stage or slow decline so long as there are still enough nutrients.

Question 30

Name 5 factors that may prevent exponential growth in a culture of bacteria, and for each explain why they become limiting.

Answer 30

1. Nutrients available- as microorganisms multiply exponentially the nutrients are used up. The nutrient level will become insufficient to support further growth and reproduction unless more are added
2. Oxygen levels- as population rises, so does the demand for respiratory oxygen so oxygen levels can become limiting
3. Temperature- enzyme-controlled reactions withing microorganisms are affected by the temperature of the culture medium.
4. Build-up of waste- as bacterial numbers rise, the build-up of toxic material may inhibit further growth and can even poison and kill the culture
5. Change in pH- As CO2 is produced by respiration of the bacterial cells increases, the pH of the culture falls until a point where the low pH affects enzyme activity and population growth

Question 31

Describe a step by step method to investigate the effect of one factor on the growth of microorganisms.

Answer 31

1. You could set up two identical colonies in different conditions of temperature
2. Set up serial dilutions of nutrients and pH at a set temperature
3. Essential to use aseptic conditions e.g. using sterile equipment and a fresh pipette after each dilution
4. Need to measure the number of microorganisms at the beginning and end of investigations.
5. Carry out serial dilutions of the original culture broth until, when you culture a given volume of the broth on your agar plate you can count the number of colonies.
6. Multiply the number of colonies by the dilution factor to give you a total viable cell count per volume for the original colony.
7. Number of bacteria= number of colonies * dilution of sample.

Question 32

Define batch fermentation

Answer 32

1. An industrial fermentation that runs for a set time
2. The microorganisms are inoculated into a fixed volume of medium
3. As growth takes place, nutrients are used up and both new biomass and waste products build up
4. As the culture reaches the stationary phase, overall growth ceases but during this phase the microorganisms often carry out biochemical changes to form the desired end products
5. The process is stopped before the death phase and the products harvested. Te whole system is cleaned, sterilised and a new batch culture set up

Question 33

Define continuous fermentation

Answer 33

1. An industrial fermentation where culture broth is removed continuously and more nutrient medium is added
2. Microorganisms are inoculated into sterile nutrient medium and start to grow
3. Sterile nutrient medium is added continually to the culture once it reaches the exponential point of growth
4. Culture broth is continually removed- the medium, waste products, microorganisms and product- keeping the culture volume in the bioreactor constant.

Question 34

Compare the advantages and disadvantages of batch and continuous culture.

Answer 34

1. Growth rate-
   a) Batch- decreases as time goes on as nutrients become limiting factor and have to have lag phase each time
   b) Continuous- even growth rate as always enough nutrients and only have one lag phase
2. Ease of setup/ maintenance-
   a) Batch- easy to maintain
   b) Continuous- more difficult to maintain
3. Consequence of contamination-
   a) Batch- Only messes up one batch
   b) Messes up the large volumes. Cost to unplanned restarting of whole culture
4. Efficiency (rate of production)-
   a) Batch- less efficient, fermenter not in use at all time
   b) Continuous- More efficient, continuous operation
5. Good for secondary or primary metabolites
   a) Batch- secondary
   b) Continuous- primary

Question 35

Define metabolite

Answer 35

A chemical produced by a cells metabolism

Question 36

Define primary metabolite

Answer 36

Are substances produced by an organism as part of its normal growth e.g. amino acids, proteins, enzymes

Question 37

Define secondary metabolite

Answer 37

Substances produced by an organism that are not part of its normal growth. They may be chemicals that an organism produces when competition in an environment becomes intense. e.g. antibiotics

Question 38

Draw a graph to show how the production of primary and secondary metabolites changes over the time of the standard growth curve.

Answer 38

1. Primary are usually formed in the period of active growth- follow growth curve
2. Secondary tend to be formed during the stationary phase of the life of the culture once the cell mass has reached its maximum.- start as reaches stationary phase and increases rapidly, goes above normal curve and then plateaus out

Question 39

Explain why the production of primary metabolites in a microorganism matches the overall growth of the microorganism population.

Answer 39

1. They are formed during the normal growth phase

2. So when the bacteria is growing primary metabolites are being produced at the same rate

Question 40

Suggest why secondary metabolites such as antibiotics are only produced after the main growth phase of a microorganism.

Answer 40

1. Needed for competition e.g. toxic chemicals plants produce to protect themselves
2. During main growth phase there isn’t competition as there is not lack of resources so they don’t need to be produced

Question 41

Draw and label a diagram of a large-scale industrial fermenter.

Answer 41

1. Look in book p600 and memorise it

2. Too much to explain

Question 42

Explain how an industrial fermenter can manipulate the growing conditions inside it.

Answer 42

1. It has probes for temperature/ pH/ oxygen concentration
2. Has cooling jackets to change temp
3. Has acid/base inlets to maintain optimum pH
4. Has pressure gauge with waste gas outlet- let excess gas out- CO2 produced in respiration
5. Compressed air inlet with filter and sparger- Distributes oxygen through the liquid- lots of tiny bubbles so SA:Vol ratio is large
6. Nutrient/ inoculent inlet with filter- maintain nutrients needed and filter reduces chance of contamination

Question 43

Explain why temperature needs to be carefully controlled in an industrial fermenter.

Answer 43

1. If too low microorganisms will not grow quickly enough
2. If too high enzymes start to denature and the microorganisms are inhibited or destroyed.
3. Bioreactors often have a heating/ cooling system linked to temperature sensors and a negative feedback system to maintain optimum conditions.

Question 44

Explain why type and time of addition of nutrient and

oxygen concentration need to be carefully controlled in an industrial fermenter.

Answer 44

1. Oxygen and nutrient medium can be added in controlled amounts to the broth when probes or sample test indicate that levels are dropping
2. Needed to maintain growth of microorganisms

Question 45

Describe why fermenters are stirred

Answer 45

1. Inside the bioreactor are large volumes of liquid, which may be quite thick and viscous due to growth of microorganisms.
2. Simple diffusion is not enough to ensure that all the microorganisms receive enough food and oxygen or that the whole mixture is kept at the temperature.
3. So most bioreactors have a mixing mechanism and many are stirred continuously

Question 46

Describe why fermenters need asepsis and how they are made to be aseptic

Answer 46

1. If a bioprocess is contaminated by microorganisms from the air or from workers, it can seriously affect the yield
2. To solve this bioreactors are sealed, aseptic units.
3. Have steam inlets to input steam to sterilise- turns to liquid water so doesn’t need to be cleaned out

Question 47

Explain why isolated enzymes may be preferred to the use of whole organisms in some biotechnological processes.

Answer 47

1. Less wasteful- whole microorganisms use up substrate growing and reproducing, producing biomass rather than product. Isolated enzymes don’t
2. More efficient- Isolated enzymes work at much higher concentrations than is possible when they are part of the whole microorganism
3. More specific- No unwanted enzymes present, so no wasteful side reactions take place
4. Maximise efficiency- Isolated enzymes can be given ideal conditions for maximum product formation, which may differ from those needed for the growth of the whole microorganism
5. Less downstream processing- Pure product is produced by isolated enzymes. Whole microorganisms give a variety of products in the final broth, making isolation of the desired product more difficult and therefore more expensive.

Question 48

Define downstream processing

Answer 48

Downstream processing refers to the recovery and purification of biosynthetic products, particularly pharmaceuticals, from natural sources such as animal or plant tissue or fermentation broth.

Question 49

Explain why extracellular enzymes tend to be used more than intracellular enzymes in biotechnology.

Answer 49

1. Extracellular enzymes are secreted, making them easy to isolate and use
2. Each microorganism produces relatively few extracellular enzymes, making it easy to identify and isolate the required enzyme. Microorganisms produce hundreds of intracellular enzymes which would need extracting from the cell and separating
3. Extracellular enzymes tend to be much more robust than intracellular enzymes. Condition outside a cell are less tightly controlled than conditions in the cytoplasm, so extracellular enzymes are adapted to cope with greater variations in temp and pH than intracellular enzymes

Question 50

Describe the advantages of using immobilised enzymes.

Answer 50

1. Immobilised enzymes can be reused - which is cheaper
2. Easily separated from the reactants and products of the reaction they are catalysing so reduced downstream processing- cheaper
3. More reliable- high degree of control over the process as the insoluble support provides a stable microenvironment for the immobilised enzymes
4. Greater temperature tolerance- immobilised enzymes are less easily denatured by heat and work at optimum levels over a much wider range of temperatures, making the bioreactor less expensive to run
5. Ease of manipulation- The catalytic properties of immobilised enzymes can be altered to fit a particular process more easily than those of free enzymes e.g glucose isomerase can be used continuously for over 1000 hours at high temps which keeps running costs low.

Question 51

Describe the disadvantages of using immobilised enzymes.

Answer 51

1. Reduced efficiency- the process of immobilising enzymes may reduce its activity rate
2. Higher initial costs of materials- immobilised enzymes are more expensive than free enzymes or microorganisms. But unlike free enzymes and microorganisms they don’t need to be replaced frequently
3. Higher initial costs of bioreactor- the system needed to use immobilised enzymes is different from traditional fermenters so there is an initial investment cost
4. More technical issues- Reactors which use immobilised enzymes are more complex than simple fermenters- more things which can go wrong

Question 52

State and describe the 4 ways in which enzymes can be immobilised.

Answer 52

1. Surface immobilisation- adsorption to inorganic carriers
2. Surface immobilisation- covalent or ionic bonding to inorganic carriers
3. Entrapment- In matrix
4. Entrapment- membrane entrapment in microcapsules or behind a semi-permeable membrane

Question 53

State which immobilisation methods may affect the shape of an enzyme’s active site and explain why.

Answer 53

1. Surface immobilisation

2. Adsorption

Question 54

State which immobilisation methods may affect the rate at which substrate molecules can gain access to the active sites of the enzymes and explain why.

Answer 54

1. Entrapment in matrix

2. Entrapment in membrane separation

Question 55

Describe the advantages and disadvantages of adsorption surface immobilisation in which enzymes can be immobilised.

Answer 55

Advantages
1. Simple and cheap
2. Can be used with many different processes
3. Enzymes very accessible to substrate and their activity is virtually unchanged
Disadvantages
1. Enzymes can be lost form matrix relatively easily- weak bonds

Question 56

Describe the advantages and disadvantages of covalent/ionic bonding surface immobilisation in which enzymes can be immobilised.

Answer 56

Advantages
1. Costs varies
2. Enzymes strongly bound and therefore unlikely to be lost
3. Enzymes very accessible to substrate
4. pH and substrate concentration often have little effect on enzyme activity
Disadvantages
1. Cost varies
2. Active site may be modified in process

Question 57

Describe the advantages and disadvantages of entrapment in matrix in which enzymes can be immobilised.

Answer 57

Advantages
1. Widely applicable to different processes
Disadvantages
1. May be expensive
2. Can be difficult to entrap
3. Diffusion of substrate to and product from the active site can be slow and hold up the reaction
4. Effect of entrapment on enzyme activity very variable, depending on matrix

Question 58

Describe the advantages and disadvantages of entrapment- Membrane separation in which enzymes can be immobilised.

Answer 58

Advantages
1. Relatively simple to do
2. Relatively small effect on enzyme activity
3. Widely applicable to different processes
Disadvantages
1. Relatively expensive
2. Diffusion of substrate to and product from active site can be slow and hold up reaction

Question 59

Describe 6 examples of the use of immobilised enzymes in biotechnology.

Answer 59

1. Immobilised penicillin acylase used to make semi- synthetic penicillins from naturally produced penicillins. Useful for treating infections caused by bacterial resistant to original penicillin
2. Immobilised glucose isomerase used to produce fructose from glucose.
3. Immobilised lactase used to produce lactose-free milk. Hydrolyses lactose to glucose and galactose.
4. Immobilised aminoacylase used to produce pure samples of L-amino acids used in in the production of pharmaceuticals, organic chemicals, cosmetics and food
5. Immobilised glucoamylase used to complete breakdown of starch to glucose syrup. Amylase breaks down starch into dextrins. Glucoamylase breakdown dextrins to glucose
6. Immobilised- nitrile hydratase- produces acrylamide which is a very important compound used in the production of plastics