Microbial Lifestyles and Metabolism (Dr. Kreft): How Microbes Grow

Question 1

Biomass and cell number can be used as measurements of?

Answer 1

growth

Question 2

Explain different ways biomass can be a measurement of growth!

Answer 2

• Protein assay ( 50% biomass is protein and does not change much)
• Dry mass (dry washed cells and weigh them)
• Optical density (OD)
  L> particles scatter light, bacteria are particles. OD is the measure of biomass rather than number of cells. Measure of cell number only so far as numbers correlate with mass (balanced growth)

Question 3

Explain different ways cell number can be a measurement of growth!

Answer 3

• total count: count cells in known volume under the microscope (counting chamber) -> cells/mL
• Viable count - CFU (colony forming units) - spread 0.1mL of (diluted) culture on agar plate and count # of colonies forming …CFU/mL

Question 4

Of the various ways to measure growth what is the most convenient method?

Answer 4

• Optical Density (OD)
• If OD is too high (>0.5) have to dilute sample as response is no longer linear
• OD proportional to biomass per volume
• OD not proportional to cell number per volume
• Cell number correlates with biomass only if the cell size is constant i.e. in balanced growth but not in stationary phase

Question 5

What are the two types of systems you can have for growing microbes?

Answer 5

• open system

- closed system - no addition of substrates during growth or can be closed off to exposure to environment

Question 6

What are the four growth phases in batch culture?

Answer 6

1. Lag
2. Exponential
3. Stationary
4. Death

Question 7

Explain the lag growth phase!

Answer 7

• conditions change from pure culture to new one. Microbes respond to this change - takes time to adapt therefore growth rate does not start strong right away. Gene expression changes. Specific growth rate will not increase until exp phase.

Question 8

Explain the exponential growth phase!

Answer 8

• composition of cells does not change. Just growth. High cell density increase. Substrate can run out quickly form high to low. Examine oxygen consumption here?

Question 9

Explain the Stationary growth phase!

Answer 9

• oxygen is used up quickly etc…enter a shock since substrate is gone. Gene expression changes to adapt. Use up internal resources

Question 10

Explain the death growth phase!

Answer 10

• death is exponential after some transition. All internal resources are used up

Question 11

Growth yield =??

Answer 11

• biomass formed per substrate consumed

Question 12

Is a continuous culture (chemostat) an open or closed system?

Answer 12

• Open: resources enter and biomass and left over resources leave
• structure: sterile air or other gas + fresh medium from reservoir via flow rate regulator—-> culture
  L> gaseous headspace + culture vessel
  L> waste products etc leave via overflow and effluent containing microbial cells as well.

Question 13

Microchemostat?

Answer 13

• microfluidics allows the study of single cells growing in a chemostat

Question 14

Steady state?

Answer 14

• if state of the system does not change with time (all variables are constant)
  low dilution rate (D) (20% of maximal specific growth rate)
  ** increase= decrease
  ** specific growth rate = dilution rate u(S) = D
  L> obtained at a certain substrate concentration S

Question 15

Steady state : high dilution rate? what happens with biomass and substrate over time?

Answer 15

• high D (80% of maximal specific growth rate)
• biomass increases and plateaus
• substrate gradually decreases…as it falls biomass plateaus

Question 16

After running many steady states at different D we can plot these steady states as a function of?

Answer 16

• dilution rate

- it is the parameter we can control

Question 17

What happens to the graph when you plot specific growth rate versus substrate concentration? (steady states)

Answer 17

• Monod kinetics (michaelis menten enzyme kinetics)

- we control D forcing the bacteria to grow at that specific growth rate

Question 18

Ks??

Answer 18

• substrate affinity, the substrate concentration at which half Umax is reached….the lower the Ks, the higher the affinity and the steeper the increase

Question 19

Why should we use a chemostat?

Answer 19

• can keep many cells under constant conditions - ideal for systems biology and physiology
• can study dependence of growth on substrate concentration (monod kinetics, yield not quite constant due to maintenance energy, cells growing at different rates differ in ribosome content, gene regulation)

Question 20

Is the chemostat more like the natural environment vs batch culture?

Answer 20

• yes
  -open system reaches steady state unless perturbed, slow growth rates in chemostats and nature, low substrate consecration in chemostat and nature, can enrich bacteria from nature which have high substrate affinity
  -

Question 21

What are two huge areas of study, chemostats can be used for?

Answer 21

• Evolution: experimental evolution in the lab

- Ecology: competitive exclusion principle

Question 22

Explain growth yield !

Answer 22

• total biomass formed/ total substrate consumed
• total boomers formed = biomass in stationary phase - biomass at beginning = delta X= Xt-X0
• total substrate consumed = substrate at the beginning - substrate left in stationary phase aka delta S= S0-St
• Growth Yield –> Ys= delta X/ delta S
• **The more substrate consumed, the more biomass formed…growth field as the ratio remains constant