Lecture 5: Flashcards
What is a major environmental factor controlling microbial growth?
temperature
Cardinal temperatures
the minimum, optimum, and maximum temperatures at which an organism grows
Optimum
- where organism grows FASTEST (according to temp)
- you can give it the temp it likes most (optimal temp), but if nutrient [ ]’s are not on point, pH isn’t controlled, salt [ ] isn’t good etc. it doesn’t matter if you give optimal temp
- but it’s just 1 variable - more needs to be considered for the reality
enzymatic reactions occurring at maximal possible rate
These cardinal temperatures are characteristic of…
each different organism
Minimum temp
mem. specifically solidified
- LESS mobile “sleeping” –> metabolically inactive
- membrane gelling; transport processes so slow that growth cannot occur
Maximum temp
mem. melted
- higher temp
- death
- breakage of VdW’s b/c of increased movement
- protein denaturation; collapse of the PM thermal lysis
If membrane is melted what has happened to its structure and what does it mean abt the cells state
falls apart - completely dismembers - no shape so cell dies b/c PM all about shape (butter in microwave)
- CELL DIES ABOVE MAX GROWTH TEMP
When membrane is solid has the cell lost its membrane integrity or is it just less mobile?
less mobile –> “sleeping”
- metabolically inactive but if you put it at high temps it will become active again (this is why your old not to freeze thaw things again and again)
Min to optimum temp if characterized by what on the graph?
very gradual slope temp decrease doesn’t have a dramatic effect as temp increase
What will mem. state be at its optimum (necessary state for PM)?
semi-fluid
- best growth rate possible
Optimum to max we see…
substantial drops in the growth rate that occur within that region
What will happen to the movement & therefore VdW’s interactions within the PL tails at excessively high temps?
breakage of VdW’s b/c of increase movement
-melted membrane; puddle that loses function and loses structural integrity
What will happen to the movement & therefore VdW’s interactions within the PL tails at cold temps?
membrane phospholipids move LESS in COLD allowing MORE van der waals to form –> “gelling” or solidification
- but mem’s need to be semifluid
Describe what happens to the protein (has to have certain shape) when HEAT is added vs when its in COLD temps
a protein needs to have a certain shape to function properly
when HEAT is added breaks H-bonds, VdW’s & ionic bonds DENATURED = non-functional
when COLD is added move LESS & ionic, H-bonds & VdW’s form DENATURED - COMPACT b/c increase # of bonds
- lil easier to recover from
- functional for cold loving organisms
Which is more lethal high or cold temps?
HIGH TEMPS= DEATH
How can microorganisms be classified into different groups name the groups?
Microorganisms can be classified into groups by their growth temperature optima
- Psychrophile
- Mesophile
- Thermophile
- Hyperthermophile
Psychrophile:
low temperature
phile= friend in greek
Mesophile:
midrange temperature
- include us & medically relevant bacteria & flora within the body
- biggest group
Thermophile:
high temperature
Hyperthermophile:
very high temperature
Where can mesophiles be found?
organisms that have midrange temperature optima (~38 degrees celsius)
• Warm-blooded animals (include us, cows, goats etc.)
- endothermic organisms that have bacteria living in them
• Terrestrial and aquatic environments
- diff bodies of water
• Temperate and tropical latitudes
- warm-temp within this range most of the time
ex- e.coli
Cold-Loving Microorganisms
- Extremophiles
- Psychrophiles
- Psychrotolerant
Extremophiles
• Organisms that grow under very hot or very cold conditions
* prefer very extreme temps
OR like LOW pH/HIGH pH/HIGH [salt] etc.
Psychrophiles
• Organisms with cold temperature optima (<20oC)
- friends of cold temps
• Inhabit permanently cold environments
- Deep ocean, Arctic and Antarctic environments
ex- polarmonas vasculota
Psychotolerant
- Organisms that can grow at 0oC but have optima of 20oC to 40oC
- More widely distributed in nature than true psychrophiles
can tolerate - don’t have a desire in either temps (can tolerate either), but prefer temps that are warmer (mediocre like we like)
List 2 ways a mem. can offset freezing to maintain semi-fluid state & do characteristics of life even when its super cold?
- MORE UNsaturated PL’s tails - so VdW’s are less likely to form even though they want
- Produce ANTI-FREEZE compounds - prevent water from solidifying
- mem. stay in fluid state that’s req & inside the cell as well
Describe some Molecular adaptations that support psychrophily
(present inside cell that allow for support)
- if they don’t have those molecular dets, there will def be problems with their ability to thrive
• Production of enzymes that function optimally in the cold
- if it can’t handle these cold conditions then its out of luck
• Modified cytoplasmic membranes
- High unsaturated fatty acid content
Explain how unsaturated FA tail prevent solidification?
Unsaturation creates a kink in that fatty acid tail -
makes packaging challenging
- positioning within the mem.
(could also be both tails)
- prevents bonds from forming even though there’s less movement
As things move MORE & MORE, bonds…
BREAK
As things move LESS & LESS, bonds…
FORM
Above ~65oC…
only prokaryotic life forms exist
(NO EUK CAN) bc cell has so many organelles, so complex, more you have the more things that can go wrong
Above ~65oC what types of metabolic species are present
• Chemoorganotrophic (use organic molecules) and chemolithotrophic (use inorganic molecules) species are present
• No phototrophy above approx. 70oC - chemotrophy
- phototrophic organisms have a bunch of intracellular mem. content that allows for the absorption of light energy & conversion into chemical energy (therefore, that machinery can’t handle high temp, it loses its structural integrity & ability to function & that machinery to be able to convert light energy into chemical energy also has temp restrictions, even if we’re talking about a photosynthetic prok (cyanobacterium)
• High prokaryotic diversity
- even at excessively high temps - therefore high density translates into fact that living in these high temps, some organisms use nitrogen metabolism, others use sulfur metabolism, diverse in terms of where they live & enzymes & wastes they produce etc. that translate into what these organisms will look like when they’re growing
- Both Archaea and Bacteria are represented (proks, but at extreme temps its mostly archaea)
Thermophiles:
organisms with growth temperature optima between 45oC and 80oC (more mediocre than hyperthermophiles)
• Terrestrial hot springs, very active compost –> nutrient rich, therefore more metabolism, more to eat, means more diversity which means more metabolic activity, which produces heat as a waste product
- fact its so metabolically rich due to all the nutrients, that’ll produce a ton of heat selectively for organisms that like higher temp
Hyperthermophiles:
organisms with optima greater than 80oC
• Inhabit hot environments, including boiling hot springs and seafloor hydrothermal vents that can experience temperatures in excess of 100oC
(breaking H-bonds holding molecules together & you get phase change to gaseous state - water remains liquid - imp. b/c water is a polar solvent & so it must be liquid for cell activity)
• Current temperature maximum record is held by an archaeon, Methanopyrus kandleri, which can grow at 122oC
- wouldn’t be destroyed by an autoclave
What happens to H20 when it reaches 100?
its boiling
break h bonds holding molecules together and have a phase change to gas
Explain how h20 remains liquid in hydrothermal vents?
b/c of pressurization of h20 even though it boils at this temp under this high level of pressure the h20 molecules are forced into close proximity to each other even though heat wouldn’t want them to mive more but bc they are so close they form H bonds and stay in a liquid state
Autoclave
used to sterilize material
combines temp & pressure so water remains as steam @ 121 degrees celsius (necessary to achieve sterilization)
destruction of all life, viruses & ENDOSPORES (can tolerate envir extremes)
Sterilize definition
destruction of all life, viruses & ENDOSPORES (can tolerate envir extremes)
Where should you check for antibiotics?
check for antibiotics in a place you know that temp & envir is v. unique, microbial diversity will be characteristic of that place, so what might this organism be producing that might be of use to us
Are antibiotics we find made in the lab
No they usually are from other living organisms they are not things you go to lab and make you could but you will usually fail
Describe what molecular adaptations thermophiles have
- Specific modifications provide thermal stability to enzymes and proteins
- Modifications in cytoplasmic membranes to ensure heat stability
- Bacteria have lipids rich in saturated fatty acids
- Archaea have lipid monolayer rather than bilayer
Describe why thermophilic bacteria have saturated FA?
no kinks- closer together if you increase temp you will want to break vdws but saturation allows for them to stay together
What do archaea that are thermophiles have in CM?
Archaea have lipid monolayer rather than bilayer (space = no VdWs there)
Hyperthermophiles produce…
enzymes widely used in industrial microbiology
- Example: Taq polymerase used to automate the repetitive steps in the polymerase chain reaction (PCR) technique
- Hydrolytic enzymes including proteases, cellulases and lipases
Taq polymerase
used to automate the repetitive steps in the polymerase chain reaction (PCR) technique
- we use a thermal stable enzyme b/c we would never be able to seperate the 2 strands outside the cell without an enzyme and if we used normal poly it would create a lot of destruction?
Enzymes of thermophiles are…
more stable (b/c functioning under excessive characteristics) and tend to have higher activity than their mesophilic counterparts
*rxn rates can be increased by increased temperature
* in our cell we would never do this b/c too destructive and if you turn up temp you turn up all reactions
whereas enzyme will be able to turn up a few and not all
What are the upper temperature limits for life?
- New species of thermophiles and hyperthermophiles are still being discovered
- Laboratory experiments with biomolecules suggest 140–150°C (may be able to isolate an organism - imp. b/c you might run into lifeforms at high temps like hot springs, allows us to understand more about complexes that these organisms are able to generate & more about if theres antibiotics there that would’ve been ignored)
Describe the cap or limit where bacteria & archaea can grow
bacteria start at around 60 degrees celsius & cap at around 100 degrees celsius
archaea start at around 80 degrees celsius & cap at around 125 degrees celsius (therefore higher ceiling & floor –> higher temp limits)
Hyperthermophiles may be the closest descendants of…
ancient microbes
Hyperthermophiles may be the closest descendants of ancient microbes
Explain
- Hyperthermophilic Archaea and Bacteria are found on the deepest, shortest branches of the phylogenetic tree
- The oxidation of H2 is common to many hyperthermophiles
- May have been the first energy-yielding metabolism
Evolution and Hyperthermophily
Describe
Thermophilic Phototrophy - until 73 degrees celsius
Thermophilic Chemoorganotrophy (use organic chemicals) - until 110 degrees celsius
Thermophilic Chemolithotrophy (use inorganic chemicals) - until 122 degrees celsius
all cross over until 73 degrees celsius
at 95 degrees celsius - elemental sulfur & reduced iron is used for nutrients
H2 - energy poor but abundant - used to support organisms like prok. that have v. low energy demands (considered on early earth as a potential nutrient source for the earliest of life forms)
Elemental sulfur, Reduced Iron & H2 are restricted energy availability vs. organic chemicals (b/c heavily reduced) BUT can you list 2 things that would make this not a problem?
1) prokaryotes are small
2) eat more (eat more H2)- H gas was considered a potential nutrient source for potential life forms
The pH of an environment greatly affects…
microbial
Some organisms have evolved to grow best at…
low or high ph
we like 7.35 pH
neutrophiles
Most organisms grow best between pH
6 and 8
* medically relevant bacteria are present here
* can cause problems inside of our bodies
Acidophiles
Organisms that grow best at low pH (<6
Alkaliphiles
Organisms that grow best at high pH (>9)
What does pH affect?
affects protein lipid structure
As you change the pH, it has dramatic effects on…
chemical structure and ability/ inability to form bonds which is associated with shape and function of a protein
ex; COOH at 2.2 = protonated cant form bond
above 2.2 = deprotonated= can form ionic bodn
NH3+ @ ph 9.6= protonated can form bond
ph above can’t form bond= deportonated
how can we manipulate organisms
by using pH extremes
- put lemon juice on chicken acidity changes protein structure tenderizes it
acidity kills and microbes
Give an example of how in the human body pH protects us?
vaginal pH= 4.5 protect against STI sperm is protected by alkalinity of semen so it doesn’t get destroyed and only things that are supposed to be ar e
The bottom line in the different adaptations is that
• The cytoplasmic membrane maintains its integrity at the growth pH
• The internal pH of a cell must stay relatively close to neutral even though the
external pH is highly acidic or basic
Explain how cell maintains a neutral ph
If the cell is in a low pH environment the environment has a high concentration of protons and these protons will want to move into the cell due to rule of diffusion (high to low) this isnt good bc ir will acidify the internal environment so it needs to have elaborate mechanisms to pump out acid if acidophile bc it has lots of protons outside that will want to come inside
What do microbial cultures contain?
Microbial culture media typically contain buffers to maintain constant pH- its your responsibilty as the experimenter to add a buffer so the organism isnt restricted by pH and can grow indefinetly
ex- lots of lactose in cartoon of milk and microbes can do lactic acid fermentation and as they ferment lactose they start to build up acid which limits their growth
buffers
neutralize acid or base produced as metabolic waste
Some bacteria produce….
- Some bacteria produce acids
- Acetic, lactic, sulfuric acid –> decreases the pH
- Some bacteria grow on amino acids
- Releases ammonia —> increases the pH
the amnio groups pinks up a proton and decrease inn acidity bc you buffered protons and ph increased and solution becomes more basic
Water activity (aw):
water availability; expressed in physical terms
• Defined as the ratio of vapor pressure of air in equilibrium with a
substance or solution to the vapor pressure of pure water
• Reflects the amount of water that is interacting with ions and polar compounds in solution
*HOW MUCH H20 IS AVAILABLE FOR CELL TO USE
What type of solvent is water explain?
water is a polar solvent
partial +/- charge provides favourable interaction with ions and polar monomeric units
can provide solubility and keep things in solution
this is why our blood is mainly h20 so it can keep nutrients in solution and all ions that are critical for nervous system and muscular function
Typically where is the solute concentration higher
the cytoplasm has a higher solute concentration than the surrounding environment
• Water will want to move into the cell creating turgor pressure- water rushing in might cause cell to swell and burst depending on how much water
- cell will have lots of solute in small volume so high concentration= valid for prokaryotic cells especially
environment= hypotonic, less solute cytoplasm= hypertonic, more solute
What happens when cell is in an ENVIRONMENT with a higher external solute concentration?
water will flow out
environment= high solute hypertonic
cell= hypotonic low solute
cell shrivels and dehydrates
Our cell is _____% H20
60- 80%
Our blood/ECF/cytoplasm/most cytoplasm has a ___% NaCl =
0.9%- isotonic
Halophiles:
grow best at reduced water potential;
have a specific requirement for NaCl
• Many marine microbes
- cell will lose lots of water due to highly concentrated environment
Extreme halophiles:
: Require high levels of NaCl
for growth
• 15 – 30%
* 30x concentration in our cells, extreme tendency for normal cells to lose water loss and dehydrate
* this cell has the capacity to water and have mechanism to prevent this
• Ex) Microbes from Great Salt lake or the Dead Sea
Halotolerant:
can tolerate some reduction in
water activity of environment but generally grow
best at lower solute concentrations
• Ex) Staphylococcus aureus- would prefer things were normal but if salty external environment thats okay not as reproductively successful but no dramatic effects
- Lives on human skin
- Grows best at low NaCl
- But can tolerate up to 17.5%
Are these optimal conditions according to the growth rate with respect to NaCl the only condition that you need to consider when you discuss the organism & its ability to reach its max growth?
No you have to look at temp pH nutrients
• Osmophiles
Organisms that grow with high sugar as solute
Xerophiles
• Organisms able to grow in very dry environment
* high sugar very low water, there must be accomidations b/c cells usually like rly high water
Specialized and rare organisms
- Honey, jams and jellies do not have many organisms growing in them
- Beef jerky and salted cod
this is b/c honey can be stores without refrigeration b/c its very hypertonic water is lost to the surroundings and the bacteria aren’t able to grow
* same can be said for any medically relevant bacteria
High osmolarity created with NaCl (EC) is used to select for…
s used to select for acid producing
microorganisms
• Used for sauerkraut and pickle fermentation
• Combination of high salt and low pH prevents the growth of most pathogens in the completed product
- cell will want to dehydrate but cell can handle it and metabolically produce acid lower pH of surrounding outcome is organism is collectively controlling growth of PH
What are mechanisms for combating low water activity?
increasing the internal solute concentration by:
• Pumping inorganic ions from environment into cell- inorganic doenst mean lactic acid
- pump inorganic ions inside making cytoplasm more isotonic making effort to hold water inside the cell
• Synthesizing or concentrating organic solutes
• Compatible solutes: compounds used by cell to counteract low water activity in
surrounding environment
Compatible solutes:
: compounds used by cell to counteract low water activity in surrounding environment
* not going to upset function of the cell
lots of ions you bring in upset concentration gradients so they aren’t compatible have to make sure whatever you bring into the cell to offset loss of water will not upset something else
Obligate aerobes
require oxygen to live
if no O2 cant survive
Strict anaerobes
do not require oxygen and may even be killed by
exposure
Facultative aerobes/ anaerobes
can live with or without oxygen, they use oxygen
when it is available
* aren’t killed by oxygen but prefer to use it to get a higher energy yield
Aerotolerant anaerobes
can tolerate oxygen and grow in its presence
even though they cannot use it
* NO ETC-ONLY FERMENTATION
• Microaerophiles
can use oxygen only when it is present at levels
reduced from that in air
- poisoned by it satisy themselves at low concentrations
thioglycolate broth
complex medium; reducing medium that converts oxygen into a reduced end product
oxic zone
coloured production in medium indicates presence of oxygen
anoxic zone
coloured production indicates absence of O2
O2 is very non polar so it will naturally be kicking around on gas that is at the top of the medium
will have a hard time diffusing thru bc growth medium is liquid broth
Inoculate with bacterium of interest
Obligate anaerobe
poisoned by O2
- is at bottom of tube
- do anaerobic respiration rather than fermentation to get higher energy yeild
Inoculate with bacterium of interest
Obligate aerobe
at top- oxic zone
not poisoned by o2 require it- aerobic respiration
Inoculate with bacterium of interest
Aerotolerant
equal dist- not competing for spots at top or bottom
- not poisoned by O2
- dont have a ETC
- cant use O2 meaning they cant do anaerobic respiration bc no ETC so have to do fermentation
Inoculate with bacterium of interest
Microaerophile
just right below oxic zone
poisoned by O2 but need it for cell respiration
What would happen if Microaerophile organisms (right below oxic zone) localize itself a lil bit higher in the test tube?
they would die bc O2 concentration inside of the cell would accumulate cause poisonous effects and cells wouldn’t survive
Inoculate with bacterium of interest
Facultative aerobe
concentrated throughout tube but prefer the oxic zone prefer oxygen but can switch muscles are faculattive not poisoned use cell respiration or fermentation
How can anaerobic organisms be grown?
Special techniques are needed to grow anaerobic microbes
- Reducing agents (take Oz add e- to make H20 which is benign) may be added to culture media to reduce oxygen
- Thioglycolate (create anaerobic enviroment in test tube) , cysteine, H2S and other sulfur containing compounds
- Removal of air, and replacement with an inert gas
- Ex) nitrogen or argon
Catalase
H202 + H202
Perioxdase
H202 + NADH + H+
Superioxide dismutase
O2 + O2-
Super oxide catalse combo
O2
Superoxide reductase
O2 + rubrodoxin —> H202
