Living systems Flashcards
what is biotechnology
the use of biological processes to create useful products
What is the process of creating a biotech product from raw materials?
- Raw material upstream processing fermentation downstream processing
examples of biotech products
wine, cheese, vinegar, insulin and citric acid
red biotech?
the pharmaceutical branch which uses bacteria to produce drugs e.g. disease preventation
White biotech?
- White biotech is the aid of living organisms and enzymes e.g. biofuels to biodiseal
Green biotech?
- Green biotech is the use of agriculture such as plants less suspectible to pests e.g. water purification
What is cell growth
essential element of fermentation for the growth of a organism
Growth is an orderly increase of…
cellular constituents
what does optimising the environment for growth require?
balancing the economic feasibility of maintaining a suitable chem environment
What does growth depend on?
the ability to form new protoplasm from the nutrients available
What does cell growth involve
An increase in the cell mass and number of ribosomes and cell division
Methods of measurement of cell mass
- Direct physical measurement of weight after centrifugation
- Direct chemical measurement of chemical component
- Indirect measurement of chemical activity
- Turbidity measurements to determine amount of light scattered by suspension of cells
- Microscopic count
- Viable cell count
Lag phase
after inoculation of cells the population remains unchanged so no cell division occurs but the metabolic activity may increase
expo phase
cells divide by binary fission at constant rate
stationary phase
exhaustion of available nutrients and space growth stops
death phase
population declines
What is a closed system
It is a batch culture where there is a lack of input and output of materials once batch medium has been inoculated, a discontinous process which environment and organisms are changing continually
What is an open system
It is a continous culture which is capable of obtaining steady state where there is balanced input growth substrate and ouput of organisms
equation for increase in cell mass
ln x = ln x0 + n ln 2
*n= t/td, ie no of generations = total time/doubling time
equation for increase in cell mass in terms of specific growth rate
ln x = ln x0 + µt
or 2=exp(ut) doubling
t=generation time
What is a growth limiting substrate
nutrient medium containing one limiting substrate at low concentrations
what limits cell synthesis
nutrient concentration in culture vesssel
Monod kinetics eq for specific growth rate
µ = µmax . s/(Ks + s)
Lineweaver burke plot
slope?
x intercept?
y intercept?
ks/umax
-1/ks
1/umax
plot is /u v 1/s
what is cell yield coefficent
amount of cell mass produced per unit amount of substrate consumed
Yield factor eq
Yfg= - deltaF/deltaG
f= moles produced G= moles consumed
observed biomass yield
Y’xs=-DeltaX/deltaSG
X=biomass produced
SG= mass of substrate
what is a continuous culture
a continuous feed of influent solution containing substrate and nutrients and a drain of effluent solution containing cells and metabolites
what is a chemostat
allows control of rate of growth which can be used to optimise the production of products
What is a primary metabolite
produced at high flow/dilutuion rate stimulating cell growth
What is a secondary metabolite
produced at low flow/dilution maintaining cell numbers
What two parameters are controlled in a chemostat and why?
Dilution rate and influent substrate conc. By controlling the dilution rate we can control the specific growth rate. By controlling the substrate conc we can control number of cells produced or cell yield in chemostat
dx/dt=?
ux-Dx
u=d is steady state
u>D utilization of substrate exceeds supply so slow growth
U
critical dilution eq
Dc=Umax(s/(s+Ks))
Why do we have critical dilution
when a chemostat is operating at Dc , if the dilution rate is increased further, the growth rate will not be able to increase (since it is already at µmax ) to offset the increase in dilution rate.
what is maintence energy
the level of energy required to maintain a cell
purpose of a fermentor
control temp and ph
reduce evapouration
minimise the use of labour
maximise computer control
scales of operation for a fermentor/bioreactor
laboratory 20L
pilot plant 5000L
production 20,000L
Most important factor in fermentor design
height to diameter ratio
why do we implement impellers in bioreactors
to reduce bubble size hence increase surface area for oxygen transfer
What is genetic engineering
the direct manipulation of an organisms genes using biotechnology to change the genetic make up if cells
what is metabolic engineering
the direct improvement of cellular properites through direct modifications of biochem reactions
what are plasmids
self replicating pieces of DNA
what is gene cloning
genes are inserted into a plasmid then added to bacteria for replication
Steps of gene cloning
- Isolate the plasmid
- Isolate gene of interest
- Insert the gene into the plasmid
- Bacteria will take it through transformation
- Cells divide along with the plasmid and form a clone of cells
Why is bacteria grown in a culture
to produce copies of the isolated gene of interest
what is Metabolism
the summation of chemical reactions in a organism
what are autotrophs
organisms that use co2 as their sole carbon
what are chemotrophs
life forms that obtain their energy by injecting carbon
Phototrophs
i.e. plants via sun
Chemotrophs
oxidation of co2 and lipids
anabolism
construction path
Catabolism and how it works
degradation path, it is the breakdown of macromolecules into building blocks then the oxidation of building blocks to produce acetyl coA which is further oxidised to co2 and water
gycolysis under aerobic conditions
pyruvate further oxidises and enters the krebs cycle
gycolysis under anaerobic conditions
pyruvate is converted into a reduced end product
lactate in muscle) = homolactic fermentation. In yeast = alcoholic fermentation yield ethanol and CO2
As cells have limited quantities of NAD+ what should happen?
it must be recycled after reduction to NADH
what happens to NAD+ under aerobic conditions
oxidised in mitochondria
what happens to NAD+ under anaerobic conditions
NADH is replenished by reduction of pyruvate
what is pathway flux
Is the rate at which input metabolites are processed to form output metabolites
what are enzymes?
catalysts that preform a wide variety of biochemical reactions in the cell
How do enzymes differ from chemical catalysts
they catalyse under mild conditions
have a high degree of specificity
enzyme activity is regulated in the cell
oxidases
oxidation reduction
what enzyme involves the transfer of function groups
Transferases
what enzyme involves the hydrolysis of reactions
hydrolysis
lyases
group elimination of double bonds
isomerases
isomerisation
what enzyme involves bond formation
ligases
what is catalytic potential equal to
enzyme activity
how do enzymes bind to substrates
through a series of non covalent bonds
how is enzyme activity measured
substrate depletion, product production and can also be measured by coupling with an enzymatic reaction to others which transforms the product into a more detectable analyte
enzyme catalysed reaction
E+s–> ES–> E+P
where [s] is high enough to convert E to ES so the second step is the rate limiting step
reaction velocity eq
vo=vmax[S]/Km+S km=[S] when v0=vmax/2 enzymes with low km achieve max catalytic efficency Km varies with temp, ph and enzyme at high [S] vo approaches vmax
eadie hofstee plot parameters
V vs V/[S]
slope= -km
y-vmax
x=vmax/km
Detergent enzymes and their conditions
proteases, lipases and amylases are added to detergents where they catalyse the breakdown of chemical bonds on the addtion of water
Condtions –> ph 9-11, 60 degrees
proteases
remove protein stains such as blood, grass and egg. Hydrolyse the protein and break them down into more soluble polypeptides
Amylases
remove residue of starch foods such as mash potato
cellulose
modify the structure of cellulose fibrils such as those found on cotton
Lipases
remove fatty stains such as lipstick
detergents should be produced using?
Bacillus
what was the first cost effective detergent and what was it based on?
lipases based on the use of rDNA technology. it has made is possible to alter the dna sequence randomly or at specific sites of lipase enzyme in order to change the amino acid sequence
what properties of lipase has protein engineering improved?
substrate specificity
thermostability
proteases stability
enzyme production process
gene+plasmid–>recombinant plasmid –> bacterial transformation –> protein
steps of enzyme production
screening - choosing appropriate micro organism
modification - improving microbial stain
lab scale - determine optimum conditions
pilot plant - large scale
upstream processing
cell factory(bioreactor) sepration of cells (centurifgation) enzymes secreted from cells downstream processing
enzymes secreted in cells –> enzymes inside cells –> cell lysis –> downstream
downstream processing
LL extraction chromatography purified enzyme three by producs(solid, liquid formulation and the reaming enzyme is imbolized)
biomass -> …..->biofeul
sugars
examples of biomass
cereal crops and lignocellulose
proteins and carbs
why is biomass not compatible with existing engines
it is a solid whereas conventional feuls are gases and has a low energy density and high moisture and oxygen content
examples of biofeuls
gasoline, hydrogen gas and propane
production of bioethanol
extraction of sugar cane –> saccharification of corn –> pre treatment
(grains)milling (water and enzymes)saccharification (yeast and sugar)fermentation (mash from fermentation)distillation can be dehydrated to bioethanol
what is milling
disadvantages
mechanical crushing of cereal grains to release starch
time consuming, expensive and slow
saccharification
heating and addition of water and enzymes for conversion into fermentable sugar
fermentation
of the mash using teast converting bioethanol and c02
saccharification of starch
starch is mixed with water at 60 degrees for 5-10mins
starch dissolves in water to form mash
liquefication of mash by adding amylase at 80 degrees for 2 hours to degrade starch
saccharification of glucoamylase added at 65 degrees for 30 mins to produce glucose
common microbes in glycolysis fermentation
saccharmyces cerevisae and zymommonis mobillis
fermentation process
1) in yeast fermentation glucose solution is obtained from starch saccharification and is cooled to 32 degrees
2) yeast culture is added and glucose is metabolised to ethanol and co2
3) some of the released energy and glucose are utilised by the yeast cells to support growth the rest of the energy becomes heat to fermentation
normal inoculation ratio for fermentation
5-10% 6-8hrs
how long does active fermentation last
12 hours then fermentation activity slows down due to less glucose being available
what happens during slow fermentation
cells do not grow anymore biochem reactions are limited by substrate conc
sources for bioethanol production
corn farm in us and brazil
60% is based on sugar cane conversion
three constituents for lignocellulose
celluose, hemi-cellulose and lignin which enhances strength and compactness
what is the structure in lignocellulose like
complex structure due to strong linkages between molecules
advantages of using lignocellulose
increases available surface area
reduces particle size
pre treatment leads to solubising hemi-cellulose and increasing enzymatic hydrosybility of cellulose
biological pre treatment
fungi and bacteria slow no chemicals solubises micro-organisms brown white and soft rot fungi white and soft rot both attack cellulose and lignin but white is more effective for pre treatment
Chemical pre treatment
dilute sulfric acid can acheive high reaction rates
but there is high costs and neutralisation is required
Alkaline pre treatment
can disrupt lignin structure and decrease crystalinity
but there is less sugar degradation and naoh can be recovered
why are advanced biofeuls good
can drop into existing infrastructure because they have similar properties to petroleum but requires large biorefineries to produce high yields
what leads to corrosion
ethanol
what is the main challenge in isobutanol production
is using native hosts to convert feedstocks into advanced bio fuels to overcome regulation of biofuel pathways to improve yields.
what is isobutanol
a next generation product which builds on the foundation and provides additional solutions to various challenges not met by first generation products
advantages of isobutanol
- Isobutanol has more hydrocarbons then ethanol therefore has a better energy content and is less polar
- Absorbs less water
- Volatility
- good Phase separation
- Good platform molecule
- Good blend properties
what 3 pathways does the isobutanol pathway consist of
- Glycolysis to provide pyruvate
- Valine biosynthesis to metabolise pryruvate to KIV
- Ehrilich pathway which is required for degradation of KIV to isobutanol
why is a high activity of KDC essential is isobutanol production
it links valine metabolism and ehrilich pathway so is essential for high levels of isobutanol
How can we increase isobutanol production
by over expression of genetic engineering by increasing KDC and ADH.
Salmonella
distributed in domestic wild animals such as pigs and cattle
in humans it is generally contacted through consumption of contaminated food of animal orgin
symptoms - diarrhoea, fever and abdominal cramps
Listeria Monocybogenes
found in soil and water and animal digestive tracts
food with long shelf life under refridgeration
symptoms- gastroentritis, meningitis
onset 9-48hrs
duration
2-10 days
Campylobaceri-jeuni
distributed in warm blooded animals
symptoms - abdominal pain, diarrhoea and fever
onset 2-5 days
duration 2-10 days
Esherichia coli
found in cattle food implicated with this are uncooked hamburger, dry cure salami, yogurt, cheese made from raw milk symptoms - bloody diarhea onset - 1-8 days duration 5-10days
Staphyloccus aureus
found on skin and nose foods at high risk of transmitting toxins are those that do not cook but handle such as sliced meat and sanwiches symptoms - vomitting and diarrhoea onset 6hrs duration 24hrs
Bacillus cereus
found in rice
causes vomitting and diarrhoea
onset 8-16hrs
duration is 24hrs
Clostridium prefringens
commonly found in raw meat such as beef, poultry, gravy
symptoms - abdominal pain, stomach cramps followed by diarrhoea
onset 8-16hrs
duration 24hrs
Clastidium
anerobic bacteria so can only grow in the absence of oxygen
onset 12-72 hours
what should be considered for foods that carry food borne pathogens
paesturization such as milk and fruit juices
standard sterilisation
what eq can describe it
steam sterilisation for reactors 121 degrees for 15mins at 103kpa
arrhenius eq
Bacillus stearothermophilus
spores are commercially available for validation for steam sterilisation unit operations
number of viable organisms eq
Nt/No=exp(-kt)
sanatation
control variables
reduction of the oppourtunity for bacterial contamination by removing it from process equipment
Control variables are cleaning agent conc, cleaning solution temp, time and velocity
Need uniform flow over tank surface which is function of application rate, surface and viscocity.
what does cooking do
Cooking kills viruses, bacteria, parasites and destroys toxins. Uncooked food contains viruses, live bacteria, parasites and toxins
what happens when re-heating food
if they do not reach 70 degrees bacteria will survive and toxins remains.
corrective action
reduce impact of an outbreak
preventive action
stop it from happening again by finding where it went wrong
method for cultivation
- Rinse the seed(useful source for contamination) and remove the damaged seed
- Soak for 8hrs
- Rinse
- Grow for 3 days at 15 degrees (ideal for salmonella growth)
- Package
- Distributed
are metabolic pathways reversible or irreversible?
irreversible – highly exergonic (ΔG = -ve) so reactions go to completion. This provides the pathway with direction.
Metabolite concentrations are a function of
Thermodynamics
Reaction kinetics
