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biotechnology Flashcards

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1
Q

what is biotechnology

A

use of living organisms or parts of living organisms in industrial processes
encompasses gene technology, gene modification, selective breeding, cloning, use of enzymes in industry and immunology

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2
Q

examples of use of biotechnology in industry

A

produce food, drugs
remove toxic materials= bioremediation

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3
Q

reasons why m/o are used in biotechnology

A

no welfare issues to consider
enormous range of m/o
can be artificially manipulated by GE relatively easily e.g. human insulin
m/o dint produce unproductive cells/tissues
nutrient requirements simple and cheap
short life cycles
huge quantities produced when given growth requirements
lower temp and pressure required than if use chem engineering
not climate dependent

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4
Q

standard m/o life cycle

A

reproduce as often as every 20-30 minutes under ideal conditions

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5
Q

reasons why m/o’s are used in biotechnology: no welfare issues to consider

A

fewer ethical issues than keeping livestock (all that is needed is optimum conditions)
animals would need health checks

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6
Q

reasons why m/o’s are used in biotechnology: enormous range of m/o’s

A

capable of carrying out many different chemical reactions

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7
Q

reasons why m/o’s are used in biotechnology: do not produce unproductive cells or tissues

A

products often released so easy to harvest products purer

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8
Q

reasons why m/o’s are used in biotechnology: economic considerations (nutrient requirements are simple and cheap)

A

can feed on waste/byproducts from other industries
can be GM easily to utilise materials otherwise wasted

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9
Q

reasons why m/o’s are used in biotechnology: lower temp and pressure needed than in chem engineering

A

cheaper product
saves fuel and cuts emissions

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10
Q

reasons why m/o’s are used in biotechnology: not climate dependent

A

processes can take place anywhere in the world

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11
Q

why are fermenter conditions kept at optimum and sealed

A

optimum for growth to maximise product yield
sealed aseptic unit to avoid contamination from m/o’s from air
prevents growth of unwanted bacteria which would compete w culture m/o for nutrients and space, decreasing yield of product. may also produce toxic chemicals which may spoil product, destroy cultured m/o and products. (in food/medicine: all product must be discarded in this instance)

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12
Q

fermenter: how is pH regulated
why

A

acid/base added
pH probe to measure pH so monitored and can be maintained at optimum for enzyme activity eg respiration
prevents denaturing

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13
Q

fermenter: how is temp regulated
why

A

temperature probe measures temp for optimum enzyme activity e.g. for respiration
prevents denaturing
cooling jacket and cold-water inlet cool bc respiration releases heat

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14
Q

fermenter: what does impeller do

A

ensures nutrients evenly distributed and keeps temp even
mixes m/o

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15
Q

fermenter: what does sparger do

A

distributes O2 evenly

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16
Q

fermenter: what does compressed air do

A

sterile air, provides O2 in aerobic fermenter
enables aerobic conditions

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17
Q

fermenter: what does steam do

A

sterilisation
kills contaminating m/o’s so they cannot compete w culture m/o for nutrients and space so increased yield

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18
Q

fermenter: what does antifoam do

A

removes foam
stops it clogging pipes
so can use fermenter for longer

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19
Q

primary metabolite example

A

ethanol from Saccharomyces cerevisiae

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20
Q

when is ethanol (1ary metabolite) made and collected

A

ethanol made as pat of normal growth of the m/o (in log phase) so product curve closely matches the m/o curve
can be collected from fermenter continuously: so maintain conditions continuously

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21
Q

when is continuous fermentation used

A

when primary metabolite is the required product

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22
Q

what is a primary metabolite

A

a product synthesised in normal metabolism when the m/o is actively reproducing

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23
Q

describe continuous fermentation

A

m/o are inoculated into sterile medium and start to grow
sterile nutrient medium is continuously added to the culture once it reaches the exponential point of growth
culture broth is continually removed (medium, m/o, waste and desired products) so culture volume in fermenter is constant

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24
Q

examples of continuous fermentation

A

single cell protein (Quorn/mycoprotein)
bacteria to produce insulin)

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25
example of secondary metabolite
penicillin from Penicillium crysogenum
26
when is secondary metabolite eg penicillin made
made when m/o growth slows i.e. in stationary phase product curve doesnt match growth curve population kept in closed culture and metabolites collected at end
27
when is batch fermentation used
when secondary metabolite is required product
28
when is secondary metabolite synthesised
only shown there is limited nutrient availability in stationary phase
29
describe batch fermentation
m/o are inoculated Ito a fixed volume of sterile medium as the growth takes place, nutrients are used up and both new biomass and waste products build up as the culture reaches stationary phase overall growth ceases but during this phase the m/o often produce the desired end products as secondary metabolites process stopped before death phase and products are harvested and fermenter is emptied and sterilised and then new batch culture can be started yp
30
examples of secondary metabolites
penicillin yoghurt beer enzyme for washing powder
31
batch vs continuous production: fermenter type
B: closed C: open
32
batch vs continuous production: used to produce what type of metabolite
B: 2ary (made in stationary phase) C: 1ary (made in exponential phase)
33
batch vs continuous production: time period
B: culture grown for fixed time period C: culture grown continuously
34
batch vs continuous production: nutrients added when
B: nutrients added at beginning only C: nutrients added continuously once it reaches exponential growth
35
batch vs continuous production: when is culture broth removed
B: removed at end before death phase (waste, nutrient and product) C: culture broth removed continuously
36
batch vs continuous production: efficiency?
B: less efficient (time wasted shutting down, removing product, restarting) C: more efficient use of time bc continuous
37
batch vs continuous production: length of exponential phase
B: short- slower growth rate due to limiting factors C: long- fast growth rate maintained
38
batch vs continuous production: examples of uses
B: wine and beer, yoghurt, cheese, enzymes for washing powder, penicillin (fed-batch) C: quorn, mycoprotein, human insulin
39
batch vs continuous production: difficulty?
B: easy to set up & maintain C: can be difficult to maintain conditions so that exponential phase is maintained. foaming, clumping and blocked inlets pose problems
40
batch vs continuous production: contamination effects
B: if contamination occurs only 1 batch is wasted C: contamination can affect the volume of product/organism
41
advantages of using mycoprotein to produce food for human consumption
production of protein can be many times faster than that of animal/plant protein biomass produced has v high protein content 45-85% production can be increased/decreased according to demand no animal welfare issues m/o's provide a good source of protein protein contains no animal fat or cholesterol m/o's can easily be GM to adjust the AA content of protein SCP production could be combined w removal of waste products production is independent of seasonal variations not much land required
42
disadvantages of using mycoprotein to produce food for human consumption
some people may not want to eat fungal protein or food that has been grown on waste isolation of the protein: the m/o's are grown in huge fermenters & need to be isolated from the material on which they grow protein must be purified to ensure it is uncontaminated microbial biomass can have a high proportion of nucleic acids which must be removed AA profile may be different from traditional animal protein and particularly it can be deficient in methionine infection: the conditions needed for m/o's to grow are also ideal for pathogenic organisms. care must be taken to ensure culture not infected w wrong organisms palatability: protein doesnt have the taste or texture of traditional protein sources
43
aseptic techniques purpose
reduces likelihood of contaminating the medium with unwanted bacteria or fungi
44
why to wear clean lab coat, eye protection, tie long hair back, cover skin cuts
prevent contamination of m/o from m/o on skin
45
why wash hands
removes any bacteria/fungi from hands
46
what to disinfect working area w
1% virkon solution
47
why disinfect working area
kills any m/o on surface
48
why keep windows closed
reduce entry and movement of m/o
49
why not talk
reduce transfer of m/o's
50
why have bunsen burner on nearby to heat air
hot air rises so creates convection current to prevent airborne m/o settling creates area of sterile air
51
why pass neck of bottle over flame as you open it and close it
causes air to move out of container so stops entry of m/o's
52
how to sterilise wire inoculating loop
hold in bunsen. flame until it glows red hot heat slowly from base to tip kills m/o's
53
how to sterilise equipment
dip in ethanol and flame keep ethanol away from flame or use autoclave (e.g. pressure cooker: steams equipment at high pressure) sterilise Petri dishes using UV light
54
why not lift lid off Petri dish completely
just enough to allow introduction of desired m/o reduced chance of unwanted m/o's getting onto the agar if lid must be removed, minimise time plate is open
55
how to seal Petri dish and why
with 4 pieces of tape (not all the way round) reduces chance of contamination by airborne m/o's allows plate to be aerobic so prevents potentially dangerous anaerobic bacteria growing
56
temperature ot incubate plate at and why
20-25C not 37C (human body temp) to prevent potentially harmful bacteria growing
57
what to do w glassware and metalware before and after contact w desired m/o
pass through flame to kill any m/o's
58
each colony on a plate arises from how many bacterial cells therefore?
1 can count number of colonies to calculate an estimate of the number of viable bacterial cells (colony forming units) in the original culture
59
why is dilution plating useful
numbers of individual m/o's in a broth can be very large; this means that after pouring a sample of broth onto an agar plate and incubating it for a few days there may be too many colonies to count crowded colonies tend to grow across each other making it impossible to count accurately
60
what kind of dilution is used for dilution plating
serial dilution starting culture is diluted by a factor of 10 several times
61
which dilution plate to use why
after incubation, plates w >300 colonies or <30 colonies are discarded plates w too many colonies lead to inaccuracies plates w small numbers have greater margins of error as small changes in no. counted will lead to big changes in estimate (any anomalies have larger impact)
62
how to ensure accurate measurements of viable cell count
clean pipette w a high resolution and low uncertainty solutions must be stirred/ inverted to mix before taking next sample use aseptic techniques to prevent contamination, which will change dilution by making solution stronger/weaker than intended, and some of the colonies counted will not be from original broth so an overestimate of viable cell count
63
immobilised enzyme definition
an enzyme that is self in place and therefore NOT free to diffuse through any reaction mixture
64
how are some biotechnological processes simplified how to overcome barriers involved
taking enzymes out of microorganisms: enzymes not used up in reaction so end up in suspension w product isolating product from enzyme is expensive: overcome by immobilising enzyme so they do not mix freely w substrate
65
methods to immobilise enzymes
adsorption surface immobilisation entrapment membrane encapsulation
66
describe adsorption
enzyme bound to supporting surface by a combination of hydrophobic and ionic links
67
suitable surfaces for adsorption
clay porous carbon glass beads resin
68
describe surface immobilisation
enzymes bonded to supporting surface e.g. clay using covalent or ionic bonds enzymes bonded using cross-lining agent which may also link them in a chain
69
describe entrapment
enzymes trapped in a matrix e.g. polysaccharides (cellulose mesh) that does not allow free movement calcium alginate beads often used in school
70
describe membrane encapsulation
encapsulation in microcapsules made of a semi-permeable membrane (membrane separation)
71
example of immobilised enzyme
immobilised enzyme in alginate beads lactase convertes lactose to glucose and galactose by hydrolysis can be used to provide lactose free milk
72
advantages of adsorption
cheap to do bound w AS exposed so accessible to substrate can be used for many different processes
73
disadvantages of adsorption
AS may be distorted by these additional interactive changes to 3ary structure, so may decrease enzyme activity enzymes can be lost from the matrix (surface) relatively easily
74
advantage of surface immobilisation
due to strong bonds, enzyme is much less likely to become detached and leak into the reaction mixture (compared to adsorption)
75
disadvantages of surface immobilisation
more expensive than adsorption bonding can distort AS, decreasing enzyme activity
76
advantages of entrapment
enzymes unaffected by entrapment enzymes protected by matrix so optimum conditions maintained
77
disadvantages of entrapment
substrate molecules must diffuse matrix and products have to diffuse out enzyme AS arranged irregularly only suitable for small substrate and products
78
advantages of membrane encapsulation
relatively simple technique relatively small effect on enzyme activity
79
disadvantages of membrane encapsulation
costs more than entrapment substrate and products must be small enough to diffuse through membrane diffusion can be slow so hold up the process
80
advantages of using immobilised enzymes
enzymes don't mix with the product, so extraction costs are lower enzymes can be reused a continuous process is made easier, as there are no cells requiring nutrients, reproducing and releasing waste products enzymes are surrounded by the immobilising matrix, which protects them from extreme conditions- so higher temps or a wider pH range can be used w/o causing denaturing
81
disadvantages of using immobilised enzymes
immobilisation requires time, materials, specialist training, and equipment so setup costs are higher immobilised enzymes can be less active bc don't mix freely w substrate and less access to AS so less ESCs formed contamination means whole system would need to be stopped and so is expensive to deal w
82
milk passed through bioreactor still has lactose present; how to remedy this
lactose would be broken down into glucose and galactose need reduced flow rate need to repeat the process/run milk through again
83
explain why a continuous culture method wouldn't be suitable for the manufacture of penicillin
2ary metabolite made when m/o kept short of nutrients (stationary phase) remains in log phase
84
suggest why limited amounts of glucose are added at regular intervals to the culture medium the fed-batch process
to keep culture alive to provide respiratory substrate maintains culture in stationary phase/prevents rapid growth
85

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