Microbial growth kinetics, isolation, preservation & maintenance of industrial microbes

Question 1

3 setting for applied microbiology

Answer 1

Disease related : infection control, chemotherapy

Environmental

Industrial: Food/ Beverage, pharmaceutical and genetic engineering

Question 2

Define microbes

Answer 2

• Unicellular, microscopic size/ invisible to naked eye
• Exist in single-celled form/ colony of cells
• Example: Viruses, bacteria, cyanobacteria, algae, fungi like mold, yeast, protozoa, larvae and egg forms of Helminths.

Question 3

Importance of microbes to human

Answer 3

Causes diseases (medical doctors find the symptoms, diagnosis and treatment according to the microbe that is causing disease)
Involve in fermentation, used industrially

Question 4

Microbial growth definition

Answer 4

Microbes grow, multiply by cell division and die.
Microbial growth is the result of both cell division and change in cell size.
Growth can happen in variety of physical, chemical and biological changes.
It is also the conversion of nutrients into biochemical compounds which are used as a energy source to product energy and biosynthesis. It is also called autocatalysis.

Question 5

What is autocatalysis in microbes

Answer 5

It is also the conversion of nutrients into biochemical compounds which are used as a energy source to product energy and biosynthesis. (self-sustaining)

Question 6

What are the 4 phases of microbial growth

Answer 6

-Lag phase
- Log growth phase
-Stationary phase
-Death phase

Question 7

Lag phase description

Answer 7

Lag time is defined as the initial period in the life of a bacterial population when cells are adjusting to a new environment before starting exponential growth.

Occurs when cells are first introduced into fresh culture
medium
- NO increase in cell number
- Period of adaptation of cells to a new environment
- NO change in number, but an increase in mass

 Cell division does not commence straight away
 Cell may need to synthesize components such as ATP,
ribosomes and essential cofactors
 Medium change may require new enzymes to be synthesized to use different nutrients
 Cells may be injured and need to recover

Question 8

Log phase description

Answer 8

It is a growth period of a cluster of cells in a culture medium. During this phase, there is an exponential increase in the number indicated by a section of the growth curve.

• Growth rate is higher
• Increase in cell mass and cell number with time exponentially
• Exponential phase
  Period of balanced growth, in which all the components of cell grow at the same rate
• Composition of biomass remains constant.

Question 9

At which phase require new inoculation of microbes into new culture medium

Answer 9

Lag phase

Question 10

What is diauxic growth

Answer 10

Multiple lag phases may be observed due to multiple source of carbon or nutrients.

Question 11

What does length of the lag phase depend on?

Answer 11

Characteristics of microbial species
Media conditions

Question 11

What does length of the lag phase depend on?

Answer 11

Characteristics of microbial species
Media conditions

Question 12

Why log phase a straight line?

Answer 12

Growth and division occurring at the maximum possible rate given the existing conditions.
Bacteria grown at binary fission.
– Rate of growth is constant; doubling at regular intervals
– exponential growth = balanced growth
• Balanced growth - cell components are manufactured at constant rates
relative to each other
– Unbalanced growth can occur if nutrient levels or environmental
conditions change
• Unbalanced growth – rates of synthesis of cell components vary
relative to one another until a new balanced state is reached

Question 13

Stationary phase description

Answer 13

Growth rate= Death rate
Net growth is zero
Even though net growth is zero, cells are metabolically active and product secondary metabolites

In closed systems population growth eventually ceases
and the growth curve becomes horizontal.

 For bacteria this is usually when the population reaches
around 109 cells per ml
 Total number of viable cells remains constant
 Balance between cell division and cell death
 Population may cease to divide but remain metabolically
active

Could occur for many reasons:
 Nutrient limitation
 Oxygen limitation
 Accumulation of toxic waste products
 Critical population level is reached

Question 14

Death phase description

Answer 14

Number of cells multiplying = number of cells dying

Death Phase (Senescence)

 Was originally thought to be simply the build up of
toxic end products resulting in the loss of viability

 Now another suggestion :
 Viable but Nonculturable (VBNC)
 Cells become dormant without changes in morphology
 Could resume growth if conditions change

Question 15

Describe what involves in Basic Research Microbiology

Answer 15

By kind of organism: Microbial taxonomy, Bacteriology, Physiology, Mycology, Protozoology, Parasitology and Virology.
By Process: Microbial metabolism, genetics amd ecology.
In relation to disease: Immunology, Epidemiology and Etiology.

Question 16

Briefly Describe microbial kinetics in Batch culture

Answer 16

Culture system containing a limited amount of nutrient, which is inoculated with the microorganism.
• Cells grow until some component is exhausted or until the environment changes so as to inhibit growth.

Question 17

What is a deceleration phase

Answer 17

The exponential phase is followed by deceleration phase,
period of unbalanced growth.

• • In this phase, the growth decelerates due to either
depletion of one or more essential nutrients or the
accumulation of toxic by products of growth

Question 18

Growth of the population is studied by analyzing the growth curve of a microbial culture grown in a closed system.

What is a closed system?

Answer 18

– No fresh medium added
– Nutrients decline and wastes
increase

Question 19

What are sigma factors

Answer 19

Bacterial protein transcription factors that facilitate promoter recognition by RNA polymerase, thus enabling gene transcription ultimately, gene expression. Each RNA polymerase molecule consists of one sigma factor and a core factor (consisting of several units). The expressiona of the genes recognised by sigma factor results in the expression of useful phenotypes that protect cells from a range of stress experienced in stationary phase.

Question 20

The Sigma Factors of Escherchia coli

Answer 20

Sigma factor 70: Housekeeping Sigma factor - recognises genes required for growth

Sigma Factor 19: The ferric Sigma factor recognizes the fec gene for iron transport.

Sigma Factor 24: Regulates and responds to extracytoplasmic functions.

Sigma Factor 28: Could of flagella and pilli synthesis

Sigma Factor 32: Controls the production of heat shock proteins.

Sigma Factor 38: Control the general stress response of cells entering the stationary phase

Sigma Factor 54: Controls the response to nitrogen limitation.

Question 21

Quorum sensing

Answer 21

quorum sensing or quorum signalling is the ability to detect and respond to cell population density by gene regulation.
Is a energy dependent transformation.
When there is a decrease in nutrients, quorum sensing is required to produce flagerium bacteria so bacteria can move during pH changes. Quorum sensing also helps in biofilm formation.

Question 22

Example of quorum sensing system

Answer 22

Signal molecule: Gamma-butyrolactones

Controlled Property:
Initiation of secondary metabolism and morphological differentiation

Taxonomic Group:
Streptomyces spp.

Question 23

Level of sigma factors controlled by what?

Answer 23

Growth rate and degree of nutrient limitation
NOT biomass concentration.

At low growth rate under nutrient depletion or toxin accumulation, sigma factors are enhanced and undergo a series of energy dependent transformations to adapt to starvation conditions before source of energy is completely depleted.

Question 24

Characteristics influenced by sigma factors

Answer 24

1. Cell size: cells are smaller in stationary phase than exponential phase: hence increasing surface area to volume ratio and facilitate the enhanced uptake of limiting nutrients.
2. production of detoxifying enzymes such as catalase and superoxide dismutase
3. repair and protection systems including DNA repair and protein protection by chaperonins
4. resistance to osmotic stress
   5.
   resistance to high temperatures
   6.
   resistance to adverse pH.

Question 25

What is programmed cell death

Answer 25

Carefully controlled process resulting in cell death that is beneficial to development and survival of the colony. It has been observed in several prokaryotes, on grown on solidified medium enabling the development of defined colonies.

Question 26

The development of streptomyces antibioticus on solidified medium involves distinct stages

Answer 26

1. Compartmentalised young mycelium (MI) develops from germinated spores.
2. Selected MI mycelium die, remaining viable segments develop into multinucleated second mycelium (MII) using substrate from dead cells.
3. MII develops in the agar medium until second PCD.
   Surviving MII synthesis outer hydrophobic layer and grow into the air at expense of dead cells.
4. The aerial mycelium produces aerial spores

Question 27

What are the classes of molecular chaperones?

Answer 27

Hsp60s, Hsp40, Hsp90s, sHsps and Hsp70.

Question 28

What are molecular chaperones?

Answer 28

Defined as the specialized proteins/ enzymes that interact with improperly folded polypeptides or partially folded, which corrects folding pathways or providing microenvironments for stabilization of folding intermediates and prevention of protein misfolding and aggregation. Act at stationary phase to prevent misfolding.

Question 29

What is osmotic stress

Answer 29

During stationary phase, toxic wastes accumulate and outside the cell(bacterium) will be increasing which will dehydrate the bacteria since water will move outside.