Week 3 - Bacterial growth and replication, including yield and responses to nutrient availability Flashcards
Energy source = light
Carbon source = carbon dioxide
= photoautotroph
• plants, algae, and cyanobacteria
• use H2O to reduce CO2, producing O2 as a side product
• photosynthetic green sulfur and purple sulfur bacteria do not use H20 nor produce O2
Energy source = chemical compounds
Carbon source = carbon dioxide
= chemoautotrophs
• hydrogen, sulfur, and nitrifying bacteria
Energy source = light
Carbon source = organic compounds
= photoheterotrophs
• green nonsulfur and purple nonsulfur bacteria
Energy source = chemical compounds
Carbon source = organic compounds
= chemoheterotrophs
• aerobic respiration - most animals, fungi, and protozoa, and many bacteria
• anaerobic respiration - some animals, protozoa, and bacteria
• fermentation - some bacteria and yeasts
Growth
increase the number of cells
Binary fission
cell division following enlargement of a cell to twice its minimum size
• duplicates that are identical
Generation time
time required for microbial cells to double in number
• depends on many factors
During cell division, each daughter cell
receives a chromosome and sufficient copies of all other cell constituents to exist as an independent cell
Reproduction in prokaryotes
- binary fission
- budding
- conidiospores (actinomycetes)
- fragmentation of filaments
Growth requirements
- physical
* chemical
Physical growth requirements
- temperature
- light (energy)
- pH
- osmotic pressure
Chemical growth requirements
• gases (CO2) -not all breathe oxygen (eg sulfur dioxide) • chemicals - organic (solids) - inorganic
Physical requirements - temperature
- maximum growth temperature
- optimum growth temperature
- minimum growth temperature
Hyperthermophiles
65 - 110 C
Thermophiles
40 - 70 C
Mesophiles
10-50 C
• environmental pathogens of humans
Psychrotrophs
0 - 30 C
Psychrophiles
-10 - 20 C
Temperatures in this range destroy most microbes, although most temperatures take more time
~62 to 130 C
Very slow bacterial growth
~52 - 62C
Rapid growth of bacteria
some may produce toxins
15 - 52 C
Many bacteria survive, some may grow
~5 - 15 C
Refrigerator temperatures
may allow slow growth of spoilage bacteria, very low pathogens
0 - 5 C
No significant growth below freezing
-30 - 0 C
Binary fission steps
- cell elongates and DNA is replicated
- cell wall and plasma membrane begin to grow inward
- cross-wall forms completely around divided DNA
- cells separate
Binary fission description
• microbial growth is the increase in number of cells, not cell size
• result of microbial growth is discrete colony - an aggregation of cells arising from single parent cell
• binary division - 1 to 2 to 4 to 8 to 16…
• divisome = when wall starts to appear
- this and proteins prime the cell for division/duplication
- peptidoglycan - for grabbing these and keeping them there
• binary fission = grows, duplicates, splits
• chorin-sensing = bacteria can sense if there’s food, temp –> replicate or not, so must communicate
Fts proteins and cell division
Fts = filamentous temperature-sensitive protein
• essential for cell division in all prokaryotes
• interact to form the divisome (cell division apparatus)
FtsZ
forms ring around center of cell
• related to tubulin
ZipA
anchor that connects FtsZ ring to cytoplasmic membrane
FtsA
helps connect FtsZ ring to membrane and also recruits other divisome proteins
• related to actin
Bacterial growth
replication usually by binary fission
(2 equal daughter cells)
• cells may not completely separate after division, leading to filaments, patches, or clusters
• some filamentous bacteria bacteria are genuine multicellular organisms
- communication and cooperation between cells
- differentiation into different cell types
- pattern formation
DNA replicates before the
FtsZ ring forms
• location of FtsZ ring is facilitated by Min proteins
- MinC, MinD, MinE
FtsK protein
mediates separation of chromosomes to daughter cells
Alternatives to binary fission
- budding
- multiple fission
- sporulation
Budding
cell splits unequally to produce a larger “mother” cell and a smaller “daughter” cell
• the mother cell may go through many rounds of replication to produce numerous daughters
• eg hyphomicrobium - a chemoorganotroph that lives on methanol and other carbon food sources
• mostly fungi
• progenitor cell produces bud that goes off to make another cell
• buds take time to mature
Multiple fission
cell becomes greatly enlarged and then divides at many points simultaneously
• eg the filamentous cyanobacterium Anabaena
• mix of budding and replication
• grows big filamentous, split –> separate
Sporulation
eg streptomyces - complex multicellular soil bacteria 1. substrate mycelium 2. aerial hypha 3. partitioning 4. spore maturation 5. dispersal 6. makes spores • spores resistant to dehydration, will germinate to form new substrate mycelia in favorable environments
Hyphae
individual organism attached into colony
• can break off and grow on its own
Requirements for successful replication
- the cell needs to grow - biosynthesis of DNA, protein, lipid, carbohydrate
- DNA replication must be completed before cell division occurs
- the chromosomes must segregate into different parts of the cell
- the septum must be formed at an appropriate point in the cell
Growth of the cell
important = biosynthesis of cytoplasmic membrane and cell wall
• gram + = lots of peptidoglycan
- live outside at first = need lots of protection
• gram - = little peptidoglycan
- live inside = don’t need a lot of protection
• gram stain separates on basis of bacterial cell wall
• penicillin attacks peptidoglycan
DNA replication and the bacterial cell cycle
DNA replication has to be tightly coordinated with cell division
1. DNA replication
2. chromosome partitioning
3. cell division
• a cell is prevented from dividing until it’s long enough, and the chromosomes have partitioned
• microbial replication = weakest part, can’t stop growth (eg contamination)
Partitioning of the chromosome copies
attachment of the chromosome to the plasma membrane is important
• DNA is replicated at a membrane-bound replication factory
• the new DNA copies remain attached to the plasma membrane
• somehow the attachment sites are pushed to opposite ends of the cell
Septum formation
the FtsZ protein (similar to tubulin in eukaryotes)
plays a crucial role in cell division
• it forms a contractile ring in themiddle of the cell
• location of the septum in the middle of the cell requires the Min proteins
Want to focus on the stage that
makes the particular product
Growth phases
- lag
- exponential
- stationary
- death
Lag phase
not duplicating, waiting for food
• food appears = exponential growth (duplication)
Stationary
waiting for food
• enclosed environment = survive then gradually die off
Replication cycle
need to known maximum growth and how to control it
• too much nutrients –> lots of waste, not utilizing at maximum rate = reduction of product produced
- so must look for optimal
Steady state
keeps going forever
• must occasionally remove bacterial load, don’t put too much nutrient in
Types of cellular transport
passive transport
active transport
Passive transport
• cell doesn't use energy • passive transport is basically absorption 1. diffusion 2. facilitated diffusion 3. osmosis
Active transport
• cell doesn't use energy • energy intake must be efficient - don't want what you're making to cost more than it makes 1. protein pumps 2. endocytosis 3. exocytosis
Chemophysical requirements - pH
- acidophiles grow in acidic environments
- molds and yeasts grow between pH 5 and 6
- most bacteria grow between pH 6.5 and 7.5
Physical requirements
osmotic pressure
• hypertonic environments, increase salt or sugar, cause plasmolysis
• extreme or obligate halophiles require high osmotic pressure
• facultative halophiles tolerate high osmotic pressure
Plasmlysed
cell membrane shrinks within cell wall (in hypertonic)
Hypertonic
- highly concentrated solution
- water molecules diffuse out of the cell
- cell shrinks
Hypotonic
lower concentration of solutes than another solution
• water molecules into cell
• cell expands
Isotonic
having the same concentration of solutes
How bacteria and plants deal with osmotic pressure
- have cell wall that prevents them from over-expanding
* in plants, the pressure exerted on the cell wall is called tugor pressure
How protists deal with osmotic pressure
- has contractile vacuoles that collect water flowing in and pump it out to prevent the from over-expanding
- eg paramecium
How water fish deal with osmotic pressure
pump salt out of their specialized gills so they don’t dehydrate
How animal cells deal with osmotic pressure
- animal cells are bathed in blood
* kidneys keep the blood isotonic by removing excess salts and water
Normal cell in isotonic solution
NaCl concentration outside cell is 0.85%
• under these conditions the osmotic pressure in the cell is equivalent to a solute concentration of 0.85% NaCl
Plasmolysed cell in hypertonic solution
NaCl is 10%
• if the concentration of solutes such as NaCl is higher in the surrounding medium than in the cell (the environment is hypertonic), water tends to leave the cell. Growth is inhibited
• plasma membrane shrinks inside the cell wall
1 colony doesn’t always represent 1 cell
so
estimate colony forming untis (CFU)
A dense broth culture could
contain 10^9 cells per ml
How do you estimate the numbers of bacteria
- look at a very small volume
- carry out a very large dilution
- find a way that doesn’t involve counting individual cells - eg using a counting chamber
Disadvantages of the counting chamber
- tedious (especially for bacteria)
- counts both live and dead cells (but this problem can now be solved using fluorescent stains that distinguish live and dead cells)
another approach = serial dilution, followed by plating on agar, then count colonies
• concentration of original culture =
dilution factor x colonies / ml plated
• if cells are in clusters then 1 cluster = 1CFU
- for high count use serial dilution
- for very low count concentrate cells by centrifugation or filtrate
Other ways to estimate cell numbers
methods that don’t rely on counting individual cells
eg optical density using a spectrophotometer
• measure light scattering which is approximately proportional to cell count
• very quick but only works on quite dense cultures
- dense bacterial cultures look cloudy due to light scattering by the cells
- this can be quantified in a spectrophotometer - measure apparent absorbance at a suitable wavelength
- this is approximately proportional to cell concentration
- need a calibration factor to calculate actual cell numbers
Spread-plate method
- sample is pipetted onto surface of agar plate (0.1 ml or less)
- sample is spread evenly over surface of agar using sterile glass spreader
- incubation
- typical spread-plate results shows surface colonies
Pour-plate method
- sample is pipetted into sterile plate
- sterile medium is added and mixed will with inoculum
- solidification and incubation
- typical pour-plate results show surface and subsurface colinies
- better than spread-plate because in spread-plate you throw away the bugs on the swab
- higher count on spread-plate = more accurate of circumstances
Grow on different mediums to find the medium that the bug grows best on
then
look at genome for chemical of interest then get genetic material out then use a different but that grows better/easier
Environmental restrictions
keep bacteria from overpopulating
With binary fission the population
doubles at each division
• under constant conditions, doubling occurs at regular intervals, giving exponential growth
Exponential growth gives a straight line if
number of cells is plotted against time on a logarithmic scale
A simple way to grow cells in the lab
batch culture - closed system
- growth medium
- inoculate with a small quantity of culture
- incubate (usually with shaking)
Batch culture instead of continuous culture because
continuous is very tedious and must always be monitored
• batch = closed, end prodcut si the end phase
Growth in a batch culture
- lag phase - inoculum adapting to new conditions
- exponential rate - population doubling at regular intervals (doubling time depends on organism, medium, and conditions)
- stationary phase - growth slows, then ceases as nutrients are exhausted and waste products accumulate
- death phase - cells start to die (starvation and accumulation of toxic waste products)
Doubling time depends on
organism, medium, and conditions
Continuous culture
open-system with a chemostat
• culture maintained in a steady-state of exponential growth
• conditions in the chemostat remain constant
- sterile air, input rate of gas to maximize organism, culture flows up to top of tube –> collect effluent
- balance stuff going in to product coming out
- adjust headspace (gas into headspace, doesn’t go right into liquid)
- what’s best for bacteria may not be best for product so keep at certain life cycle phase
Mean generation times vary widely depending on
the organism and the conditions
Vibrio natriegens
a marine bacterium found in salt marsh mud
• can double in 10 minutes
Mycobacterium leprae
a skin pathogen, the cause of leprosy
• doubling time of several weeks in vivo
E. coli in rich medium can double in
about 30 minutes
Doubling time depends on
uptake of nutrients and time required to replicate DNA, proteins, cell walls, etc
Rapid doubling gives an advantage
eg
sudden appearance of rich food
Effect of temperature on growth rate
enzymes
• extreme halophiles make acid to control environment
What limits the growth of bacteria in the wild?
eg phytoplankton - photoautotrophic microbes growing in the oceans, need only minerals, water, carbon dioxide, and sunlight
• minerals tend to be the limiting factor
(nitrate, phosphate, IRON)
• adding a small amount of iron could make a big difference to phytoplankton growth
Example of halotolerant
Staphylococcus aureus
Example of nonhalophile
E. coli
Example of halophile
Alilvibrio fischeri
Example of extreme halophile
Halobacterium salinarum
Bacteria simulate their own growth, as well as that of their neighbors, by
secreting growth factors into their environments eg Rpf (resuscitation-promoting factor)
Classification of organisms based on oxygen requirements
- aerobes
- anaerobes
- facultative anaerobes
- aerotolerant anaerobes
- microaerophiles
Aerobes
undergo aerobic respiration
Anaerobes
do not use aerobic respiration
Facultative anaerobes
can maintain life via fermentation or anaerobic respiration or by aerobic respiration
- in a tube, cells tend toward the top but are throughout
Aerotolerant aerobes
do not use aerobic metabolism but have some enzymes that detoxify oxygen’s poisonous forms
- in a tube, cells are throughout but tend toward bottom
Microarophiles
aerobes that require oxygen levels from 2-10% and have a limited ability to detoxify hydrogen peroxide and superoxide radicals
- in a tube cells in the middle of solution
Obligate aerobes
oxygen is essential
• final electron acceptor in ETC
- in a tube, all cells at top of solution
Obligate anaerobes
oxygen is deadly
- in a tube, all cells at the bottom of solution
Sergei Winogradsky
discovered chemosynthesis - the process by which organisms metabolise a number of different inorganic substrates to obtain carbon
• previously it was believed that autotrophs obtained their energy solely from light, not the oxidation of inorganic compounds such as H2S and NH4+
Winogradsky column
aerobic ^ microaerophilic ^ anaerobic ^ H2S
• gases at bottom feed up to help bugs above grow = multiorganism bioreactor
Winogradsky column - water
aerobic
• algae, cyanobacteria, aerobic heterotrophs
Winogradsky column - red-brown
microaerophilic
• purple non-sulfur photoheterotrophs
Winogradsky column - red-violet
anaerobic –> microaerophilic
• purple sulfur bacteria
Winogradsky column - green
H2S –> anaerobic
• green sulfur bacteria
Winogradsky column - sediment
H2S
• sulfate reducers, fermentative heterotrophs
Preparation of a Winogradsky column
substrates =
• cellulose
• sodium sulphate
• calcium carbonate
- the substrates are mixed into the sediment and the column is half filled with water
- the remaining half is filled with river water leaving a small air gap at the top and tightly sealed
- the column is then left for 2 months in strong sunlight
