Lecture 2 - media and growth Flashcards
How to quantify cells (cell number)
- optical density by measuring absorbance = light scatter
-> problem: cells are not always the same size so there might constructional differences, good for an indication but not for actually counting - flowcytometry
-> problem: cells are different weights, sizes etc - particle counter
- counting cells on a grid using microscope
-> problem: very slow
Problems with quantifying cells (cell mass)
living cells are 80-90% water
- water content is hard to determine accurately
Dry weight cell analysis
- first cells are centrifuged/filtered.
- then they are washed with distilled water (removing salt)
- finallt, they are dried (via heat or freezing) and then reweighed
Biomass composition: macromolecules
- lipids = esters of fatty acids
- energy storage, structure (membranes) - proteins = monomers: amino acids
- structure, catalysis (enzymes), regulation (receptors) - carbohydrates = monomers: sugars
- structure, energy storage (depends on type of organism, some store via carbohydrates, some via lipids etc) - nucelic acids = monomers: nucleotides
- genetic information, transcription (catalysis), translation
Examples of macromolecules as products (5)
- enzymes (enzymes to break down sugars etc)
- pharmaceutical proteins
- fatty acids as food supplements
- polysaccharides such as xanthan
- polyhydroxyalkanoates = bioplastics
Macro- vs microelements in microorganisms
macro = elements they need in larger amounts
- ex: C, H, O, N, P, S
micro = elements they need in smaller amounts
- ex: Mg, K, Ca, Fe, Na, Cl
Trace elements
= elements they need in even smaller amounts than microelements
- often needed for something specific
ex: Mn
- needed for every organism that grows aerobically since it is used to activate enzymes needed for that
Cmol
= the mass of the amount of a compound that contains exactly 1 mol (12 grams) of carbon
- good to use if we don’t know the exact molecular composition
What is growth media for?
It can have catabolic and anabolic pathways
- we give mos some food and they turn it into the nutrients they need
- substrate is necessary for catabolism
-> we add it for dissimilation (gives energy)
- macro-/micro-/trace elements are added for anabolism
-> we add it for assimilation, biosynthesis etc
What is desired from growth media?
we want a medium that:
- supports a high specific growth rate and/or a lot of biomass
- supports high product formation and/or high titres (yields)
-> often you only want 2 of these 4. For example, if we want a lot of cells we want to optimize to get high specific growth rate and biomass, not high product formation and high titres
it should avoid:
- high residual concentrations of nutrients
-> to avoid cost of purification and waste
- negative impact on product quality
cost of feedstocks should be in line with product pricing to be economically okay
synthetic media
a lot of different compounds in it
- usually used when working with mammalian cells, to grow them for pharmaceutical products
- should not be used when working with something cheap
minimal media
synthetic media that contains only the nutrients that are required for growth
- carbon, magnesium, sulphate, nitrogen, phosphate
complex media
a media you do not know the exact composition of
- for example, if you add yeast to then all composites of yeast will be in there as well
Could be:
- non-degradable components
- toxic components
- completely unknown compositon
- could create batch variations (different from day to day)
Positives:
- often fairly cheap
- if you don’t know what the cells need you can add yeast extract etc to see if the cells grow
prototrophic
synthesis of cell constituents from simple substrates
auxotrophic
= addition of growth factors is required
- these cells need help to grow
- ex: amino acids, vitamins, fatty acids, purines/pyimidines
- popular for genetic modification (genetic markers)
- almost always unsuitable for industrial production
examples of carbon sources for medium
- sugar (beet, cane)
- proteins (expensive => not that common)
- starch (wheat, corn, rice)
- hydrolysed starch (glucose syrups)
complex carbon sources, examples
= not only carbon sources, will provide other things as well
- molasses
- lignocellulosic materials
-> ex. cornstover, bagasse, wheatstraw
-> agricultural residues, cheap and abundant
- whey
complex carbon and nitrogen source: example and what is it used for
example: whey
- used for cheese production, animal feed, supplement of many products
- can be used as carbon and nitrogen source
- has lactose so organism needs to be able to hydrolyse lactose to use it
Nitrogen sources: minimal, examples
- ammonium
- urea
- ammonia
nitrogen sources: complex, examples
- fish meal
- yeast extract
- amino acid solution
Growth on ammonium, how does it work?
- salts or dissolved NH3
Cells take up ammonium (use charge gradient over membrane to take it in)
- ammonia is what ends up inside the cell and is used
- you are left with a proton
-> to compensate so the proteins inside the cell do not denature, the proton is transported out of the membrane
- when using ammonium as nitrogen source: titrate culture with base to couple between the growth and the addition of base to the culture
Growth on amino acids
Uptake of amino acids occur => 2 different kinds of amino acids can be formed
1. “energy forming” amino acids
2. “protein forming” amino acids
When adding to the media, what do you need to think about?
- viscosity
- saturation
- osmotic pressure
Concentration of nutrients, what do you need to think about?
- water activity, some solutes bind the water
- osmotic pressure
- influence on the cell’s metabolic control
How do mammalian cells grow?
mammalian cells and bacteria grow by fission
- cells are identical
How do yeasts and many fungi grow?
By budding.
- bud forms on the cell containing genetic information
-> when big enoug, it is cut off and a new cell is formed
- mother and daughter cells are not identical since mother cells have scars from budding
-> can bud 25-30 times before too much scarring prevents it
Fungi can still have exponential growth
- divide by branching => two branches form which then also divide => exponential growth
Cell banks - what is their function?
Cell banks are where you store the cells for research or for production
Are used for:
1. long term storage (to preserve different things so client gets the same product every time)
- identity (morphology and metabolism)
- purity (no contamination)
- suitability (viability, production stability)
- supply
- cells must be available in an appropriate amount on demand
How are cells preserved in cell banks?
- extreme reduction of temperature
- cryoprotective media
- thawing rate
- freezing media
-> thanks to protectants and cell membranes being more flexible, the crystals formed do not puncture the membranes
- dehydration
How is mold stored?
- let spores grow on rice
- add water to the rice and then you freeze dry it on glycerol
-> can then count cells and store them in cell bank
Why exponential growth does not continue forever and how to fix it
- depletion of nutrients => medium optimisation
- transfer of gases => aeration, mixing
- production of heat => cooling
- inhibition by (by-)products => improved organism/conditions)
