Week 4 - microbial growth & bioprospecting

• What is a microbial culture?

• Method of growing microbes in the lab

• Controlled environment

What are the parameters controlled in microbial cultures

• Temperature

• Nutrients (C. N, P, etc.) & water

• Agitation (mixing)

• pH

• O2

• Time (most crucial e.g. time to grow/ produce)

• How do microbes grow/replicate?

• Main mode in prokarya (bacterial & archea): Binary fission

  • Exponential growth

  • Aesexual - daughter cells are clones

• Mutations can still occur!

What are the significance of mutations and their impact on microbes

• This is a source of genetic variation

  • Often are neutral/ deleterious but sometimes can be advantageous

• Beneficial traits selected for adaptation

Describe the process of binary fission

1. Circular chromosome has an ORI - replication bubble opens and DNA polymerase begins replicating

   1. Replication fork expands

   2. Simulatenously, both the old and new strand are attached at separate points of the membrane (anchored here)

2. Growth of cell wall (new proteins) and plasma membrane (new phospholipids) between attachement points

3. Chromosomes separate

4. Fstz stimulate plasma membrane and cell wall to grow inward (starts to separate cell into two halves)

   • Fstz (filamenting temperature sensitive mutant z)

5. Two daughter cells (Ea. Have chromosome, and roughly half cytoplasm - virtually same)

6. repat cycle

Describe Fstz discover, function y & what organisms its found in

• Fstz (filamenting temperature sensitive mutant z)

function: Fstz stimulate plasma membrane and cell wall to grow inward (starts to separate cell into two halves)

discovered: in E.colu in 1960s as a temp. sensitive protein

organisms: later found to be universal across all(ish) bacteria, most archea, plant chloroplast, mitochondria (of unicellular - lesser eukaryotes)

How long does one binary fission cycle take?

• generation time (or doubling time, t_D, DT, td)

  • Time taken to double during balanced growth (in optimal conditions)

• Differ between species (even in optimal)

Why does generation time differ between species even in optimal conditions

• Complexity

  • e.g. bigger chromosome will generally take longer

• Speed of metabolic steps

  • e.g. if evolved to withstand rougher environemnt, steps will take a long time, even in optimal

• Can vary between 20minutes (e.coli) to 12-14 days (Mycobacterium leprae)

What is the growth curve?

curve that descrives microbial growth

Describe the trend/shape of the growth curve

• If no limiting factors -> exponential

• However, this is theoretical.

  • Slowly nutrients deplete, waste accumulates in medium -> cells become stressed -> slows growth/ death

• Exponential growth is temporary

What organisms follow the growth cuve?

• Growth curve universal shape in microbes; only thing that differs is time in each stage

What is the specific growth rate & symbol

• Mu - μ -> specific growth rate

  • How fast population increase per time unit

How can we calculate specific growth rate at a given time? - formula

• μ= 1/N * dN/dt

  • N = number of cells

  • Cell concentration (cells/mL) or biomass concentration (g/L)

  • dN = change in cell number

  • Dt = change in time

Explain how the formula is obtained?

• μ= 1/N * dN/dt

• How fast population increase per time unit

• dN/dt = gives rate but does not account for disparity in cell numbers

• e.g. very different for 100 cells to produce 1 vs 1 cell to produce 1

• thus divide by N

What are the three methods to measure biomass / number of cells? Describe the two basic/rare ones (process, limitations/pros) - and name the most common method.

1. Classic/traditional method:

   • Process:

     • Take aliquot of cells in flask

     • Take small known volume (e.g. 1μL)

     • Put under microscope and physically count

   • Cons: very tedious and boring (take long time)

2. Flow cytometry

   • Process:

     • Has capillary with very very small diameter (only lets one cell throught at a time)

     • Beam of light shines + counts

   • Cons: very expensive, not all have acess

3. Spectrophotometry (OD₆₀₀) (most common)

What are the pros of spectrophotometry

• Benefits:

  • Small and sits on bench

  • Cheaper

  • Quick - data given in seconds

Describe process of spectophotometry

• Measure OD₆₀₀ of sample

  • Has optical density - light wavelength of 600

  • Put sample in cuvette + machiene

  • Light goes through

    • Fresh medium is clear - all light passes

    • The more biomass, the more cloudy - greater turbidity

  • Light asborbed by fluid or scatters (optical density) + recorded by dector

  • Amount scattered gives us a OD₆₀₀ value

• Calibration curve:

  • Do experiement with known samples and measure their OD600

  • Plot the ratio of OD600:cells/mL on curve -> calibration curve

• Calculate :

  • Take OD600 of our unknown sample + find its corresponding cell number in the curve

Why is calculating cell number important in industry?

• In bioreactors/fermentation tanks need to carefully regulate and monitor conditions - this method offers a quick way

Describe the lag phase of growth curve

• No growth

• Physiological adaptation i.e. cells metabolising to prepare for growth

• Length dependent on:

  • Species

  • History of culture (e.g. if from -80C tank may take more time)

    • Preservation method

    • State of cell (is it damaged, death phase, etc.)

      

Describe the log phase of growth curve

• 
  

 

• Exponential growth here

• Maximal growth rate (if perfect optimal conditions) is μmax

  • Unique to species and conditions

• May approach it, but rarely sustained as:

  • Limting conditions of rate:

    • Grow rate limited by nutrient, toxic waste, temperature, or pH

• Biotech aims to maximise for industry

Describe Stationary phase of growth curve

• Growth rate balanced by death (net 0)

• Due to depleated essential nurtrients, waste inhibiting growth

• Normal cell functions remain

  • e.g. energy metabolism, metabolite biosynthesis

Describe death phase of growth curve

• If incubation continued, cell death exceeds growth

• Sometimes includes cell lysis

• Rate of death of often slower than rate of growth (in exponential phase)

If goal is to produce biomass quickly what phase of growth curve must be sustained?

• sustain exponential phase

• Industry cares - money

If goal is to produce primary metabolite production what phase of growth curve must be sustained?

exponential phase

Industry cares - money

If goal is to produce secondary metabolite production what phase of growth curve must be sustained? What is an example of them?

(e.g. antibiotics) -> stationary phase

Industry cares - money

• What are the types of microbial culture compositions?

1. Pure cultures

2. Mixed cultures

What are pure cultures? Why are they used?

• Single organism present

• Why are they used?

  • For specific product e.g. one metabolite or one organism

    • e.g. e.coli for insulin; l.baccilis for yakult

What are mixed cultures? Why are they used?

• Multiple organism present - community of microbes

• Why are they used?

  • For tougher jobes, several steps + complex; need more robustness

    • e.g. wastewater treatment (lecture 3)

What is required to scale up from lab to industry?

• Scale up is from flask (mLs) to bioreactor (1000sL)

• Need to control environment to maximise growth/production:

  • Volume increases dramatically making this harder

• use a bioreactor for this

What is the problem with lack of O2 delivery in industry cultures

slow growth as 99% are aerobic (may even lead to death + mean we have to restart whole process)

What is the problem with lack of mixing in industry cultures

uneven growth & production - clump

What is the problem with lack of heat removal in industry cultures

death/product loss

What is the problem with lack of waste removal in industry cultures

toxicty/death

• What is a bioreactor

• A vessel that maximises conditions for microbe

• What conditions do bioreactors control?

• O2

• Nutrients

• Temperature

• Waste

• pH

• What do bioreactors maximise?

• Maximise growth rate, product yield

• Prevent stress/death

• What are features/components of bioreactors?

• Pump

  • to add nutrients, etc. (note sometimes this is a closed system - no pump)

• Aggitation system (mix)

• Submerged aerator

  • To disperse O2

• Thermal jackets

  • Control heat (as microbes produce lots)

• Probes

  • Monitor waste, O2, pH

How is insulin produced using the bioreactors and microbes?

• What are the microbes most commonly used?

• E.coli (most common) in large bioreactors

• Can also use yeast - sacromyces (S. cerevisiae)

How is insulin produced using the bioreactors and microbes?

• What temperature is ideal for growth and production?

• Growth (exponential/log phase) - 37C

• Once enough biomass made:

• Production - 25-30C - less stressful temperature

How is insulin produced using the bioreactors and microbes?

• What are the three carbon sources we may provide, and their pros/cons?

• Glucose

  • Most common

  • In between both

  • E.coli are very adapted to using this

    • Glucose enters metabolism quickly

  • But when excess glucose, some is used for fermentation -> lactate (toxic) - death; making our product/culture dirty

• Glycerol

  • Better option for cleaner product (no lactate)

  • Takes more effort to use this for energy (as not adapted for this)

  • However, very slow

• Mollases

  • very cheap and quick (lots of sugar - bacteria love it)

  • But, very dirty product

• Choose based on resource + desired quality

  • Mollases < glucose < glycerol

How is insulin produced using the bioreactors and microbes?

• Why do we provide nitrogen, and from what sources?

• Nucleotide, amino acid production

• Sources: ammonium (NH3), ammonium salts (NH4+), yeast extract (for E.coli), peptone

How is insulin produced using the bioreactors and microbes?

• What is the ideal pH?

pH 7

How is insulin produced using the bioreactors and microbes?

• What is an inducer, and why is it needed?

• Lactose (from milk) - stimulates production of insulin

  • As cells not adapted to be producing it need to induce them

How is insulin produced using the bioreactors and microbes?

• How do we extract the insulin precursor from our cells?

• Need to extract as insulin is INSIDE the cells - need to get it into the medium to harvest

• Centrifuge -> remove supernatant (liquid) - collect biomass (collected as precipitate) -> cell lysis (need

How is insulin produced using the bioreactors and microbes?

• How do we correctly refold insulin once extracted from the cells?

• Add denaturants (e.g. urea) to unfold/solubilise protein

• Remove urea - allows them to refold correctly based on natural affinites

• Purify

• What are the expression systems that can be used in industry? Applications, benefits and cons?

• When are prokaryotes used?

  • No post-translational modifications

  • Usually refolding is required after

• Yeast

  • Can do post-translational

  • May require refolding

• Insect

  • Proper folding

  • Can do post-translational

• Mammals

  • Proper folding

  • Can do post-translational

if i need to produce Large complex proteins with post-transcriptional/translational modifications (e.g. acetylation, glycosylation, etc.)

what expression system?

eukaryotes

if i needed to produce simple proteins with no post-translational/transcriptional modifications - what expression system?

prokaryotes

Describe resources/equipment acess needed for expression systems (bacteria, yeast, mammals, insects)

• Bacteria/yeast - cheap, little acess

• Insect, mammals, are more expensive, more sophisticated equipment, greater media control

  • Mammalian cells specficially have not evolved to survive unicellularly

    • Need to impose this which is complicated + lots of resources

• What are types of cultivation strategies?

• batch

• fed-batch

• continous

Applications/example of batch culture

• Commonly preffered for beer, wines

  • Use sacromyces - s. cerevisiae

  • Product: ethanol (metabolite; not enzymes/microbe)

    • Cells die as ethanol is toxic

pros/cons of batch culture

• Advantages:

  • simple, easy to operate

  • Low contamination risk as closed system

• Limitations:

  • Nutrients become limiting

  • Waste accumulates

  • Growth eventually stops

What is batch culture

• All nutrient added at start

• Until harvest, no input or output during culture

What is fed-batch culture?

• Fresh medium slowly pumped into bioreactor from feed tank during culture

  • Volume of cell culture increases over time

    • Dilutes waste and enables prolonged exponential growth

• No effluent is removed

• Why is fed-batch effective? What are things it minimises?

• Substrate inhibition

  • Some substrates added at once in excess (e.g. methanol, ethanol, acetic acid) inhibit growth

  • Fed-batch slowly pumps in

    • Reduces inhibition as time given for nutrients to be used

    • Maintains concentration at optimal levels

    • Extends log phase

• High cell density

  • Large amounts of nutrients may be required to reach high cell density; however can become inhibitory

  • Means must gradually feed to do this

• Glucose effect

  • Undesireable byproducts may form when nutrients in high concentrations

  • e.g. baker's yeast -> if excess glucose - ferment -> ethanol (toxic)

  • When adding fresh media - dilutes waste

What are application/example of fed-batch

• Preffered for insulin, industrial enzymes, antibiotics, culturing microbes - common in industry

• e.g. detergents - industrial enzyme

  • Product: Peptidase/amylase from Baccilus sp.

pros/cons fed-batch

• Advantages:

  • Higher cell density and product yield (as exponential growth maintained)

  • Better nutrient control

• Limitations

  • Complex

  • cost

What is continous culture

• Fresh media added from feed tank

• Equal volume waste removed and pumped into effluent tank (means no waste, no death - healthier microbes)

• Occurs continously & continously harvesting

• This means, can reach a steady state

  • Note stead state NOT stationary state

  • Here, growth is steadily at umax/exponential (sustain log phase)

    • Is it at umax or just a u that is steady (former not mentioned)

distinguish between stationary state and steady state

• open system (continous batch) means, can reach a steady state

  • Note stead state NOT stationary state

  • Here, growth is steadily at umax/exponential (sustain log phase)

    • Is it at umax or just a u that is steady (former not mentioned)

pros/cons continous batch

• Advantages:

  • Constant productivity

  • Stable conditions

• Limitations

  • Difficult to control

  • Higher cost (as need to continously mointor + operate)

  • Greater contamination risk

    • Open system

    • Very expensive to scrap if contamination occurs - need to basically restart whole batch

common continous batch application example

• Vaccines, recombinant proteins, wastewater treatment (lecture 3)

  • e.g. vaccines

    • Virus part -> molecular cloning -> bioreactor -> produce antigens -> inject into patients

    • Product: recombinant protein

    • Enables large-scale + consistent production

    • By E.coli or sacromyces

compare batch, fed-batch, continous culture applications

batch: Commonly preffered for beer, wines

fed-batch :Preffered for insulin, industrial enzymes (e.g. detergents), antibiotics, culturing microbes - common in industry

continous: Vaccines, recombinant proteins, wastewater treatment (lecture 3)

• What is the bioprospecting pipeline?

1. Explore nature

2. Sample into lab - isolate and characterise different microbes

3. Screen for useful traits (testing them)

4. Cell culture & scale-up

5. Commercial product (takes years, and a very small fraction reach here)

What is bioprospecting?

• Systematic search for bioproducts with useful (financially) biological properties

  • What are some bioproducts?

    • Anything - enzymes, genes, microbes

    • Bioactive componds (molecule interacts with + produces effect on biological systems)

Why is bioprospecting important?

• Microbes can do chemistry we struggle with,

  • Antibiotics, industrial enzymes, food ingrediants, bioactive, genes for biotech

Describe Phase 1: exploring nature of bioprospecting

go into nature - take samples

• What are some locations we can search, and what things are typically found there?

• Rainforest soil

  • Antibiotics

    • High biodiversity so microbes must compete with each other

    • Developed antibiotics to kill competitors of nutrients

• Hot springs

  • Heat tolerant enzymes (e.g. Taq polymerase for PCR)

• Cold springs

  • Cold tolerant enzymes

    • e.g. for cold washes in laundary for delicate items

• Oil contaiminated soil

  • Microbes for hydrocarbon bioremediation

• Insect gut

  • One of the toughest environments for microbes

  • Biofuel enzymes (cellulose digestion)

  • Antibiotics (found in gut as again high biodiversity to outcompete)

• Salt lakes

  • Food/industrial fermentation - enzymes/microbes resistant to salt

  • e.g. for soy sauce

What is found in rainforest soil (bioprospecting)

• Antibiotics

  • High biodiversity so microbes must compete with each other

  • Developed antibiotics to kill competitors of nutrients

What is found in hot springs (bioprospecting)

• Heat tolerant enzymes (e.g. Taq polymerase for PCR)

What is found in cold springs (bioprospecting)

• Cold tolerant enzymes

  • e.g. for cold washes in laundary for delicate items

What is found in Oil contaiminated soil (bioprospecting)

• Microbes for hydrocarbon bioremediation

What is found in Insect gut (bioprospecting)

• One of the toughest environments for microbes

• Biofuel enzymes (cellulose digestion)

• Antibiotics (found in gut as again high biodiversity to outcompete)

What is found in Salt lakes (bioprospecting)

• Food/industrial fermentation - enzymes/microbes resistant to salt

• e.g. for soy sauce

Describe Phase 2: isolation & characterisation

• Take microbes into lab and culture in various conditions

  • Manipulate temperature, carbon concentration, N, P, etc.

  • Try to maximise growth

• Get a community of microbes in conditions -> isolate

• If a novel + unknown microbe is found can proceed

Describe Phase 3: screening for useful traits

• Put into various media that are selective for specific traits e.g.

• If in search of cellulases -> cellulose-rich media

  • If survive efficicently - indicates can digest cellulose

  • If sufficient levels of cellulase present can optimise for use

  • e.g. cellulase for paper industry

• If in search of proteases -> protein-rich media

  • e.g. for jean production need proteases/amylases

• If need antibiotics -> media with bacteria

  • If kill +outcompete others

In Phase 3: screening for useful traits

what would you do if in search of cellulases (+ application)

• cellulose-rich media

  • If survive efficicently - indicates can digest cellulose

  • If sufficient levels of cellulase present can optimise for use

  • e.g. cellulase for paper industry

In Phase 3: screening for useful traits

what would you do if in search of proteases (+ application)

• protein-rich media

  • e.g. for jean production need proteases/amylases

  • protein-rich media

    • indicates can digest protein

In Phase 3: screening for useful traits

what would you do if in search of antibiotics

• If need antibiotics -> media with bacteria

  • If kill +outcompete others

Describe Phase 4: culture and scale-up - what need to happen by this point

• By this point:

  1. DNA sequenced

  2. Compound of interest is characterised

  3. Computational biology - model + compare against others

e.g. test the antibiotic against various bacterial protein targets

1. Test in lab

2. Paperwork

   • For drugs takes years of test before human trials (FDA/TGA approval)

   • For industry - quicker usually

What is signficiance of phase 5: commercial product ?

• Yay you may make billions !!!

• Only small fraction arrive here

• Who does bioprospecting?

•  

  • Universities, environmental scientises, government - to understand biology, discover new molecules; study biodiversity, ecosystem function; conservation, CSIRO - manage resource sustainably

  • Pharmaceutical and biotech companies for new meds; enzymes, food, industrial product for profit

Describe antibiody timeline history for discovery

• had first discovery by alexander felmming - penicillin in 1920s

• golden era - boom of several prodced (basically all classes discovered here)

• discovery void - scientists concerned about this new drug poorly understood - implementing tests to control + basically only 1-2 classes approved

• antibiotic-resistence rise

What facilitates a new era of bioprospecting - finding a target?

omics era - helps with inspecting molecular cells

• help decide which antibiotics to use

synthetic biology - (lecture 2) - vombining CRISPR + molecular cloning + computational biology - to design new biological systems (E.g. alter metabolic pathways, combine genes) - achieve our perfect all from one microbe

AI - analyse lots of omics to predict enzyme function/ identify gene clusters/ help determine canditates to then test

• these methods reduce need to go into nature + isolate from 1000s

• instead can choose only a few to test - quicker

What facilitates a new era of bioprospecting - culturing the unculturable?

iChip - isolation chip - discovered 2009-10

cannot grow 99% microbes in lab - as so finely tuned to specific environment

collect environmental sample, dilute + pipette onto chip (has several microscopic chambers - only one cell per chamber), put back into environment (where it can grow)

can therefore culture

Describe a discovery iChip has made

• teixobactin (one of the only new class of antibiotics in 2015)

• active against several gram-positive bacteria (E.g. M. tuberculosis)

Describe ethics in bioprospecting

What is biopiracy

Descrive conventions to prevent biopiracy (bioprospecting)