Lecture 2 - media and growth

Question 1

How to quantify cells (cell number)

Answer 1

• 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

Question 2

Problems with quantifying cells (cell mass)

Answer 2

living cells are 80-90% water
- water content is hard to determine accurately

Question 3

Dry weight cell analysis

Answer 3

• 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

Question 4

Biomass composition: macromolecules

Answer 4

1. lipids = esters of fatty acids
   - energy storage, structure (membranes)
2. proteins = monomers: amino acids
   - structure, catalysis (enzymes), regulation (receptors)
3. carbohydrates = monomers: sugars
   - structure, energy storage (depends on type of organism, some store via carbohydrates, some via lipids etc)
4. nucelic acids = monomers: nucleotides
   - genetic information, transcription (catalysis), translation

Question 5

Examples of macromolecules as products (5)

Answer 5

1. enzymes (enzymes to break down sugars etc)
2. pharmaceutical proteins
3. fatty acids as food supplements
4. polysaccharides such as xanthan
5. polyhydroxyalkanoates = bioplastics

Question 6

Macro- vs microelements in microorganisms

Answer 6

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

Question 7

Trace elements

Answer 7

= 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

Question 8

Cmol

Answer 8

= 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

Question 9

What is growth media for?

Answer 9

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

Question 10

What is desired from growth media?

Answer 10

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

Question 11

synthetic media

Answer 11

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

Question 12

minimal media

Answer 12

synthetic media that contains only the nutrients that are required for growth
- carbon, magnesium, sulphate, nitrogen, phosphate

Question 13

complex media

Answer 13

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

Question 14

prototrophic

Answer 14

synthesis of cell constituents from simple substrates

Question 15

auxotrophic

Answer 15

= 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

Question 16

examples of carbon sources for medium

Answer 16

• sugar (beet, cane)
• proteins (expensive => not that common)
• starch (wheat, corn, rice)
• hydrolysed starch (glucose syrups)

Question 17

complex carbon sources, examples

Answer 17

= not only carbon sources, will provide other things as well
- molasses
- lignocellulosic materials
-> ex. cornstover, bagasse, wheatstraw
-> agricultural residues, cheap and abundant
- whey

Question 18

complex carbon and nitrogen source: example and what is it used for

Answer 18

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

Question 19

Nitrogen sources: minimal, examples

Answer 19

• ammonium
• urea
• ammonia

Question 20

nitrogen sources: complex, examples

Answer 20

• fish meal
• yeast extract
• amino acid solution

Question 21

Growth on ammonium, how does it work?

Answer 21

• 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

Question 22

Growth on amino acids

Answer 22

Uptake of amino acids occur => 2 different kinds of amino acids can be formed
1. “energy forming” amino acids
2. “protein forming” amino acids

Question 23

When adding to the media, what do you need to think about?

Answer 23

• viscosity
• saturation
• osmotic pressure

Question 24

Concentration of nutrients, what do you need to think about?

Answer 24

• water activity, some solutes bind the water
• osmotic pressure
• influence on the cell’s metabolic control

Question 25

How do mammalian cells grow?

Answer 25

mammalian cells and bacteria grow by fission
- cells are identical

Question 26

How do yeasts and many fungi grow?

Answer 26

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

Question 27

Cell banks - what is their function?

Answer 27

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)

2. supply
   - cells must be available in an appropriate amount on demand

Question 28

How are cells preserved in cell banks?

Answer 28

1. 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

Question 29

How is mold stored?

Answer 29

• 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

Question 30

Why exponential growth does not continue forever and how to fix it

Answer 30

1. depletion of nutrients => medium optimisation
2. transfer of gases => aeration, mixing
3. production of heat => cooling
4. inhibition by (by-)products => improved organism/conditions)