biotechnology Flashcards
what is biotechnology
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
examples of use of biotechnology in industry
produce food, drugs
remove toxic materials= bioremediation
reasons why m/o are used in biotechnology
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
standard m/o life cycle
reproduce as often as every 20-30 minutes under ideal conditions
reasons why m/o’s are used in biotechnology: no welfare issues to consider
fewer ethical issues than keeping livestock (all that is needed is optimum conditions)
animals would need health checks
reasons why m/o’s are used in biotechnology: enormous range of m/o’s
capable of carrying out many different chemical reactions
reasons why m/o’s are used in biotechnology: do not produce unproductive cells or tissues
products often released so easy to harvest products purer
reasons why m/o’s are used in biotechnology: economic considerations (nutrient requirements are simple and cheap)
can feed on waste/byproducts from other industries
can be GM easily to utilise materials otherwise wasted
reasons why m/o’s are used in biotechnology: lower temp and pressure needed than in chem engineering
cheaper product
saves fuel and cuts emissions
reasons why m/o’s are used in biotechnology: not climate dependent
processes can take place anywhere in the world
why are fermenter conditions kept at optimum and sealed
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)
fermenter: how is pH regulated
why
acid/base added
pH probe to measure pH so monitored and can be maintained at optimum for enzyme activity eg respiration
prevents denaturing
fermenter: how is temp regulated
why
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
fermenter: what does impeller do
ensures nutrients evenly distributed and keeps temp even
mixes m/o
fermenter: what does sparger do
distributes O2 evenly
fermenter: what does compressed air do
sterile air, provides O2 in aerobic fermenter
enables aerobic conditions
fermenter: what does steam do
sterilisation
kills contaminating m/o’s so they cannot compete w culture m/o for nutrients and space so increased yield
fermenter: what does antifoam do
removes foam
stops it clogging pipes
so can use fermenter for longer
primary metabolite example
ethanol from Saccharomyces cerevisiae
when is ethanol (1ary metabolite) made and collected
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
when is continuous fermentation used
when primary metabolite is the required product
what is a primary metabolite
a product synthesised in normal metabolism when the m/o is actively reproducing
describe continuous fermentation
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
examples of continuous fermentation
single cell protein (Quorn/mycoprotein)
bacteria to produce insulin)
example of secondary metabolite
penicillin from Penicillium crysogenum
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
when is batch fermentation used
when secondary metabolite is required product
when is secondary metabolite synthesised
only shown there is limited nutrient availability in stationary phase
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
examples of secondary metabolites
penicillin
yoghurt
beer
enzyme for washing powder
batch vs continuous production: fermenter type
B: closed
C: open
batch vs continuous production: used to produce what type of metabolite
B: 2ary (made in stationary phase)
C: 1ary (made in exponential phase)
batch vs continuous production: time period
B: culture grown for fixed time period
C: culture grown continuously
batch vs continuous production: nutrients added when
B: nutrients added at beginning only
C: nutrients added continuously once it reaches exponential growth
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
batch vs continuous production: efficiency?
B: less efficient (time wasted shutting down, removing product, restarting)
C: more efficient use of time bc continuous
batch vs continuous production: length of exponential phase
B: short- slower growth rate due to limiting factors
C: long- fast growth rate maintained
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
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
batch vs continuous production: contamination effects
B: if contamination occurs only 1 batch is wasted
C: contamination can affect the volume of product/organism
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
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
aseptic techniques purpose
reduces likelihood of contaminating the medium with unwanted bacteria or fungi
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
why wash hands
removes any bacteria/fungi from hands
what to disinfect working area w
1% virkon solution
why disinfect working area
kills any m/o on surface
why keep windows closed
reduce entry and movement of m/o
why not talk
reduce transfer of m/o’s
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
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
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
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
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
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
temperature ot incubate plate at and why
20-25C
not 37C (human body temp) to prevent potentially harmful bacteria growing
what to do w glassware and metalware before and after contact w desired m/o
pass through flame to kill any m/o’s
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
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
what kind of dilution is used for dilution plating
serial dilution
starting culture is diluted by a factor of 10 several times
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)
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
immobilised enzyme definition
an enzyme that is self in place and therefore NOT free to diffuse through any reaction mixture
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
methods to immobilise enzymes
adsorption
surface immobilisation
entrapment
membrane encapsulation
describe adsorption
enzyme bound to supporting surface by a combination of hydrophobic and ionic links
suitable surfaces for adsorption
clay
porous carbon
glass beads
resin
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
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
describe membrane encapsulation
encapsulation in microcapsules made of a semi-permeable membrane (membrane separation)
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
advantages of adsorption
cheap to do
bound w AS exposed so accessible to substrate
can be used for many different processes
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
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)
disadvantages of surface immobilisation
more expensive than adsorption
bonding can distort AS, decreasing enzyme activity
advantages of entrapment
enzymes unaffected by entrapment
enzymes protected by matrix so optimum conditions maintained
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
advantages of membrane encapsulation
relatively simple technique
relatively small effect on enzyme activity
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
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
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
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
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
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
