Lecture #3 - Microbial Growth & Nutrition Flashcards
Macronutrients
- Elements required in LARGE amounts to build macromolecules (b/c you req. a lot of it)
- The building blocks of cell material
think: screw to build house is a macronutrient b/c you need A LOT of it to build the house
How much of dry weight do Macronutrients makeup?
C,H,O,N,P and S makeup >90% of the dry weight of the cell
What does “dry weight of the cell” mean?
dehydrated - means that water is 60-80% of the weight of cell will skew the #’s too much so you consider cell in dehydrated state to see how the atoms will be req.
Protein–C,H,O,N(andS)
• Polymer of made of building blocks – amino acids
How much of dry weight do Protein–C,H,O,N(andS) makeup?
> 50% of cell dry weight
- b/c protein is the workhouse of the cell - big deal in so many things, therefore cell must have adequate amounts
Protein–C,H,O,N(andS); which are from where?
C,H,O,N - backbone of an AA
S - 2/20 AA’s (cysteine/methionine)
Lipids–C,H,O(andP)
- Building blocks = fatty acids and glycerol
- Ex) Phospholipids
- mostly HYDROPHOBIC structure
- C, H - contributes to the non-polar hydrophobic character (therefore most of hydrophobic structure)
- O - (small amount) within hydroxyl groups of f.a. tails & in glycerol comp. of neck of structure
- P - specifically found in PL’s - head group comp
Carbohydrates – C, H, O (and N)
- Building blocks = sugars
- Ex. Polysaccharides and peptidoglycan
(monosaccharide (building blocks) form polysaccharides that take on specific terms like PD)
C: 1
H: 2
O: 1
(glucose C6H12O6)
Carbohydrates – C, H, O (and N)
N is a…
sugar DERIVATIVE
- not true sugar but if it has N its a derivative of a sugar
- can be there, but never part of glucose, galactose, sucrose, ribose, deoxyribose, etc.
Protein is ____% of dry weight
55
RNA is ____% of dry weight
20.5
Why is RNA such a large value of % dry weight comp. of cell?
PRE-CURSOR (transcript) to actually give you your protein which you have so much of (55%)
Nucleicacids–C,H,O,N,P
- Building blocks = nucleotides (indiv. pieces used to form DNA & RNA)
- Ex. DNA and RNA
Describe where each Nucleicacids–C,H,O,N,P are part of
C,H,O - part of sugar (ribose or deoxyribose)
N - part of nitrogenous base
P - part of phosphate group
Carbon is ___% of dry weight
50
backbone for all organic macromolecules
Hydrogen is ___% of dry weight
8.2
only forms 1 cov. bond
Sulfur is ___% of dry weight
1.8
2/20 AA’s
Selenium is ___% of dry weight
<0.01
- used to form selenocysteine (an AA MODIFICATION)
- even though its small you still need it
Other Macronutrients – inorganic ions (K, Mg, Ca, Fe)
• Often serve as metabolic CO-factors (a sidekick)
• NON-protein component required for enzyme function
- enzyme itself is a PROTEIN
- Enzymes involved in protein synthesis require K+
- Cytochromes (e- carriers) require Fe2+ (affects reduction potential, which affects e- affinity so those e- carriers will have diff. affinity (pull/desire for those e-‘s) which will correlate with amount of energy that’s released)
- Other functions:
- Mg2+ helps stabilize membranes and nucleic acids (can relieve charge repulsion on PM)
- Ca2+ helps stabilize cell walls, and plays a role in heat stability of endospores (helps make inside of endospore stable - Ca2+/dipicolinic acid)
Describe glycolysis
glucose –> glucose 6-P
glucose –>(hexokinase - protein; has cofactor of Mg2+, allowing formation of G-6P) glucose 6-P (-)ly charged
Mg2+ (NON-protein) (+)ly charged (therefore provides temporary relief for charge repulsion that (-) charges will have with 1 another
ATP –> ADP Pi comes off and goes to form G-6P
_____ of the macronutrients should be considered when making media
ALL
therefore, DON’T expect them to grow if you left out a source (think: forgot screws - key comp)
ALSO, not all organisms like the same thing - may not like concentrations b/c they are wrong
CANNOT grow every single organism in a lab, no matter how hard you try (some are too partic)
Micronutrients
• Elements required in VERY SMALL amounts (trace elements)
- doesn’t mean they are unimp. ( they are CRITICAL for their function)
- just b/c you don’t need a lot of something, doesn’t mean you can remove it & it still will lead to an unhindered life (absolutely critical you always have this material in the cell for full growth & success & viability)
• Usually serve as COFACTORS for enzymes
- provide a support (usually ionic stability)
- can’t function without its sidekick
• Ex) Mn, Zn, Co, Ni, Cu, Mo
• Se is required to make the unusual amino acid selenocysteine (derivative of cysteine AA (has S); been modified - allowing add. function/diversity)
Micronutrients
Usually serve as cofactors for enzymes
• Ex) Mn, Zn, Co, Ni, Cu, Mo
Describe Zn2+
(can’t function without its sidekick; provide a support usually ionic stability)
Zn2+ to stabilize active site of the enzyme
- anywhere you have a (-) charge with your substrate, the (+)ity can provide ionic stability or electrostatic activity
Carboxypeptidase
Why is C so abundant, & O and H are less?
C - can form 4 cov. bonds - excellent candidate to build lipids, carbs, AA’s & nucleotides b/c you can build elaborate castles (lots of diff. options on how they can be built)
O - can form 2 cov. bonds - less diversity/options
H - con form 1 cov. bond
Growth factors
- Small organic molecules REQUIRED for growth (BUT there are things the cell can sometimes make)
- IF an organism CANNOT SYNTHESIZE the growth factor, then it must be ADDED to medium to grow that microbe in the lab
IF an organism CANNOT SYNTHESIZE the growth factor, then…
it must be ADDED to medium to grow that microbe in the lab
If you have an organism that CANNOT synthesize a GF, then gets put into a growth medium that doesn’t have that GF? Will it grow?
- either they won’t grow or they’ll grow abnormally (certain pathway for ex won’t be prod.)
- depends on what it was critical for
- expect destruction of some kind
- GFs can either be made b/c organism has a recipe within its genetic makeup to actually cell for that OR it must be provided
If you have an organism who CAN synthesize the GF. Will it rather wanna make it on its own or take what you provided in the GM? You can give or not give it to him. Would he rather you give it to him or make it on its own?
- GIVE IT TO HIM –> b/c LESS energy (less time/work)
think: someone can buy you a car or you can earn it on your own
- you’ll take, but doesn’t mean you’re not capable of working for it - have opp. to do that
Three classes of growth factors:
- Amino acids
- Purines and pyrimidines
- Vitamins
Three classes of growth factors:
- Amino acids
• 20 amino acids are needed for protein synthesis
- if you look at an Aa codon chart (there’s 20), so to be able to accommodate every codon that you encounter, you would need to have a total of 20 AA’s avail. in some copy #
(lego block - 20 diff colours/shapes etc. & then you make proteins which are lego castles)
Three classes of growth factors:
- Purines and pyrimidines
- A,G,T,CandU
- Needed to make NUCLEOTIDES, building blocks of DNA and RNA
(lego blocks that come for DNA in 4 flavours & RNA in 4 flavours & then you build nucleic acids (the castle))
Three classes of growth factors:
- Vitamins
- Small molecules used to make organic COFACTORS (usually something needed by an enzyme for full function)
- NON-protein components required by some enzymes (protein)
- Ex) Nicotinic acid –> NAD+
What is the cofactor usually made of?
NON-protein
What is the enzyme usually made of?
PROTEIN
Describe which is the cofactor and which is the vitamin
Ex) Nicotinic acid –> NAD+
Nicotinic acid - vitamin
NAD+ - cofactor (since this cofactor is ORGANIC its called a COENZYME)
- imp. cofactor that needs to be present for glycolysis & Kreb’s cycle
- necessity to have it
- inability to produce energy if the enzyme doesn’t have it & cell will die
Growth factor requirements
- Many have NO growth factor requirements
- Ex) E. coli
- Addition of growth factors to medium may promote growth
- Some bacteria require many
- Ex) Leuconostoc mesenteroides requires ALL 20 amino acids, 4 purines and pyrimidines, 10 different vitamins
Describe E. coli’s growth factor requirements
NO growth factor requirements
NOT picky –> don’t have to worry; can make what they req. by themselves
- can build things - will be AUTONOMOUS - do a lot of things SOLO (CAN COOK)
Describe Leuconostoc mesenteroides growth factor requirements
require MANY
requires ALL 20 amino acids, 4 purines and pyrimidines, 10 different vitamins
PICKY - can’t make it on their own (CAN’T COOK)
Describe PABA (p-aminobenzoic acid) growth factor
some bacteria use this pathway for folic acid syn.
FUNCTION: precursor of folic acid
- necessary by bacterium to form AA & some nucleotides
- if bacterium doesn’t have folic acid, it cannot form full spectrum of nucleotides & AA’s that it req’s & can normally do on its own
Describe Sulfa drug
antibiotic
- analogs to PABA (look like PABA, but isn’t), which confuses bacterium & allows it in
- PREVENT folic acid syn. which this is present in you
- therefore, bacterium can’t make the folic acid, AA, nucleotides
- antibiotic has PERFECT selective toxicity - b/c we don’t make folic acid, we get it from our diet, therefore when we take the drug it doesn’t interfere with our own folic acid syn. b/c we don’t do our own folic acid syn
- can be resistant
Describe Biotin growth factor
used in the formation of C-C cov. bonds
FUNCTION: fatty acid biosynthesis; some CO2 fixation rxns
Describe Nicotinic acid (Niacin)
FUNCTION: precursor to NAD+
Describe Lipoic acid growth factor
- temporary carrier
- if not present, you wouldn’t advance to next process
FUNCTION: decarboxylation of pyruvate & apha-ketoglutarate
- used in intermediate step - catalyzed by pyruvate dehydrogenase
- also used in TCA cycle in the alpha-ketoglutarate rxn as a temporary e- acceptor (just there for the mech)
- if its not there, the mech for the 2 enzymes is screwed up & aren’t able to use their product which stops metabolism & can’t go into Krebs & not able to produce things to meet ATP req.
Nutrient sources usually identified by ____
ELEMENT
H,O
• No specific nutrient
• Found in H2O and organic media components
( H & O found in H20 - always part of growth med) & (glucose & alanine AA has both too)
P
- Usually provided as phosphate salt (PO43-)
- Ex) K2HPO4, KH2PO4
- REASON: usually acquired as PO43- in the environment
- In freshwater systems PO43- is often limiting
Is there an atmospheric source of P?
NO - except in select geographical locations (phosphine)
Can’t pull P from atmosphere
- therefore, restricted to soil, water & rock –> therefore, limiting, meaning cell will run out of it when building they have the rest of the other sources (N, C etc)
- can’t build ATP, nucleotides or PL’s any further for growth phase
Limiting nutrient
(what organism is restricted by)
- In relatively LOW concentration compared to other nutrients
- When it runs out, growth STOPS despite other nutrients present
What becomes limiting first & second?
N
usually P - BUT in laundry detergents they typically have P, so now you have an add. source that’s not supposed to be there as apart of ht normal cycle - so its no longer a limiting factor, outcome is you don’t run out (supports growth)
- then N
What can happen with fertilizer for ex?
if you add to much, its osmotically active, so fertilizer will dehydrate the source & grass looks burnt if you over fertilize or did it wrong
N (many possible sources)
Inorganic N
Organic N
Atmospheric N2
Inorganic N
= NO C attached
• Provided as salts (ex. KNO3 or NH4Cl)
- satisfy a K+ req at same time and the other satisfies a Cl- at same time
• Must be reduced to NH3 – used to make amino acids (-NH2)
Organic N
• Provided as N rich organic molecules (that has a C backbone) (ex. Amino acids or short peptides) - does NOT need to be reduced
AA-AA-AA –> AA AA AA
break bonds to get free AA’s (lego blocks) to get energy from breaking an ordered molecule
Atmospheric N2
- N2 is reduced to 2NH3 – Nitrogen fixation
- NH3 is used to make amino acids
• Energetically expensive (costly to break)
- when its happening at a cell setting even: enzymes avail. will be heavily depen. on ATP avail; constantly in a multistep process utilizing energy in order to get the job done
• Can only be done by some Bacteria and Archaea – NOT by eukaryotes
Even though there is 78% atm composition of N (all around us) what is the problem with N2?
triple cov. bond that is v. stable
- extrem. hard to break - setting cell to 400 degrees is not an option to keep a viable cell
- if it happens in the cell (rare to use as N source), then it NEEDS ENZYME - that provides mech to disrupt it but also utilize LARGE amounts of energy b/c its so unwilling to destabilize on own
it is abundant (but it’s like a tease) where certain organisms have capacity to make use of it
Describe 2NO3- –> N2 –> 2NH3
2NO3 - fully oxidized
N2 - intermediate redox state
2NH3 - fully reduced (e- donor - providing energy)
if you start with NO3-, you need to turn it into NH3, to actually use it to make AA
Rhizobium leguminosum is…
CAPABLE OF N-FIXATION!
- SYMBIOTIC relation b/t plant & Rhizo.
- plants let rhizo live in its root nodules with lots of space & expect rhizo to fix N for them, so the plant can build leaves & stems, protein req, head groups of PL’s need N etc. & then the plant provides free rent & some photosynthetic sugars sent down from leaves for Rhizo to supply itself with nutrients & they can let Rhizo take whatever else from soil
Enzymes for N fixation are…
poisoned by N fixation
- & it is a problem b/c photosyn. produces O2
- so they come together & form anaerobic req’s, where N fixation enzymes could be protected (& dw about N inactivation)
Inorganic S
• Provided as salts (ex. MgSO4) (Mg2+ needed to stablize mem. & SO4 provides oxygenized S)
• Must be reduced to the level of S2- – used to make amino acids
- Assimilative sulfate reduction (into an organic molecule)
Describe SO42- –> S2- –> H2S
SO42-(fully oxidized) –> S2- (intermediate redox state) –> H2S (fully reduced)
2 AA’s needs sulfur
cysteine (CH2SH)
methionine R group CH3-S-(CH2)2
- 1st AA; usually added during protein syn - but have to ensure you have enough of it to syn. cell comes from assimilative sulfate reduction
What is the H2S form (fully reduced)?
essentially, metabolic waste (could poop out reduced S that it makes as a waste product from e- transport, but in this case it assimilates it into an organic molecule
Why would it poop out the waste if it could use it for another purpose on the inside of the cell (maintains balance of interior of cell)
What’s it called if they take reduced form of S after ETC & just poop it to outside of cell?
NON-assimilative sulfate reduction
- complete waste, don’t use to build
Organic S
• (give organism) Pre-made amino acids (that has S part of framework already) (cysteine and methionine)
& collectively, lot
• Less energy to assimilate (b/c they don’t have to do the work so they can grow faster)
- now have it & can use it to build according to their needs
- amount of work they need to partic. in is substantially lower
SO42- can be used as…
a SUBSTITUTE for O2 in the ETC = ANAEROBIC RESPIRATION
- you stick S at the end, if O2 isn’t avail.
(don’t get 32 ATP like you would with aerobic, but get more than what you’ll get for fermentation which is 2 ATP)
C
• Refers to the source the majority of C in macromolecules
main component for organic material - doesn’t matter if its a bacterium, plant cell, animal cell - we all have same core req’s for C b/c its used to build sugar, protein, lipid
Organisms placed into 2 groups based on how they obtain C:
- Heterotrophs
* Autotrophs
Heterotrophs
• Use ORGANIC carbon (means its a C that’s a component of a lipid, a C that’s a comp of a carb - something that’s an organic molecule that’ll contain C in its framework)
• One or more C is REDUCED (ie. a C atom with one or more H’s)
- ex: fatty acid
• Ex) Organic acids, alcohols, carbohydrates, amino acids
- energy rich (lot of energy if you catabolize)
Autotrophs
• Use INORGANIC carbon (CO2) as their sole source of carbon
- gaseous - energetically happy
• Requires energy to ASSIMILATE (b/c v. abundant)
Taking C source & converting to a larger, more elaborate structure
Participates in:
• PHOTOSYNTHESIS
• Ex) Anabaena
can cook! => take INorganic CO2 comes from all animals doing respiration & also from fossil fuel emission, fireplaces
- therefore, ABUNDANT; so they need a source of energy to assimilate them, in order to put it into a structure like ex fatty acid (23 e-‘s - a lot of energy to be had by stripping these e-‘s off of the framework & then using them in redox rxns to generate ATP & e- transport chain)
- therefore, can cook b/c they’re gaseous -> energetically happy & disordered the way they are (our waste). Can be fixed into the structure, so energy is stored within b/c if you break it as a heterotroph, you can obtain energy
Classes of culture media:
- Defined medium
- Minimal medium
- Complex medium
Defined medium
• EXACT chemical composition is KNOWN (follow recipe)
• Useful for studying METABOLISM
- add/delete stuff for observation to see how it affects growth
REPRODUCABLE! - can use same growth media on your own from ppl who publish it
think: making pizza from scratch & adding ingredients according to recipe
Minimal medium
• A defined medium that provides the MINIMUM nutritional requirements for growth (ie. NO growth factors)
(bare min. to promote survival)
think: bread & water (not super nutritious - nothing for organism to think)
Complex medium
- EXACT chemical composition is NOT known
- Often made from MEAT or YEAST EXTRACTS
- meat: animal tissue –> made of PL’s, nucleic acid, carbs, therefore all of these are key comp. of normal living tissue, are now part of GM (but don’t know exact comp)
- extracts: mash up to create an extract that supplies a lot of GF’s
- Supply a VARIETY of GROWTH FACTORS
- Ex) T-soy broth (no agar, therefore liq) and plates (agar - causing it to be solidified)
can add BLOOD (GM) - has O2, iron, protein etc - but don’t know exact comp
think: ready made pizza (have an idea, but not exact ingredients)
Is the defined culture medium for E. coli s or liq despite fact that its defined?
liquid
Differential medium:
• Allows different bacteria to be DISTINGUISHED
- Ex) Blood agar – T-soy plate (nutrient med) + 5% sheep’s blood (allows test of hemolysis)
- Allows differentiation of hemolytic bacteria
What is hemolysis?
blood cells that split
- causes changes in discolouration
a-hemolysis
incomplete destruction of blood cells
b-hemolysis
complete destruction (of BC’s present)
g-hemolysis
NO destruction
- not capable of hemolysis; can’t break RBC’s apart
Which hemolysis (a, b or g) is growing?
ALL GROWING
Another way to differentiate, based on the metabolic activity the organism is partic. in (apart from hemolysis):
put organism on plate:
acid waste (pink)
non-acid waste (white)
also include a pH indicator in GM - therefore, any acid will react & produce a pink colour
A type of metabolism, determines what…
an organism will look like
Selective medium:
(think: only ppl with red hair can go)
- Contain ingredients that INHIBIT the growth of unwanted microbes
- Allow ONLY specific microbes to grow
- can add something toxic
- outcome: limit growth, therefore more manageable (think: doing this to soil b/c it has so much diff. microbes (enriched)
• Ex) Mannitol salt agar
Selective medium:
Ex) Mannitol salt agar
• Contains VERY HIGH SALT, so that only halotolerant
bacteria will grow
- b/c they can maintain
- rest will die under these hypertonic conditions
• Used to ISOLATE staphylococci from skin
Selective medium:
Ex) Bile Salts
are used in our intestine to emulsify/rip apart fat to make a hydrophobic molecule easier to digest
- so only organism built to tolerate intestine can do so, & non-intestinal organisms get selected out
Enriched medium:
• Supplemented with special nutrients to ENCOURAGE the
growth of FASTIDIOUS bacteria
• Complex nutrient requirements – REQUIRE MANY growth factors
• Ex) Blood agar (encourages growth), chocolate agar (agar - nutrient source) & other things in there
- then picky organisms will grow
think: fold arms until they get exactly what they want
Some can’t grow most organisms in lab regardless of blood/choco…
NON-CULTURABLE
Some can’t grow most organisms in lab regardless of blood/choco…
How is it that we identify them, if we can’t grow them?
metagenomic analysis (PCR) to check for genetics
Energy Classes of Microorganisms
Metabolism
• The SUM TOTAL of ALL of the chemical reactions that OCCUR IN a CELL
catabolic & anabolic rxns collectively form metabolism
- think: money coming in bank account & going out
- if you have to little you tap into savings –> fat stores
- if you have to much you build savings –> nutrients coming in
Energy Classes of Microorganisms
Catabolic reactions (catabolism)
• Energy-releasing metabolic reactions (e.g. fermentation, respiration)
think: paycheque
Energy Classes of Microorganisms
Anabolic reactions (anabolism)
• Energy-requiring metabolic reactions (biosynthesis)
think: payments/expenses
ex’s:
- building RNA during transcription
- building protein during translation
- building glucose during photosynthesis
What are metabolic rxns (catabolic & anabolic) are associated with?
energy gain & energy requirements b/c catabolic rxns create DISORDER
- things happen as a result of the rxn
- like to be disorder
If you have a rxn that creates DISORDER, like…
CATABOLISM then energy gets RELEASED
If you have a rxn that creates ORDER, like…
ANABOLISM, then you have to pay for that b/c it doesn’t go according to what they actually want
Energy Classes of Microorganisms
Microorganisms grouped into energy classes depending on their source of electrons and energy:
- Chemorganotrophs
- Chemolithotrophs
- Phototrophs
Every single LIVING organism can be categorized by 2 things:
- Where they get their C from
- autotrophs vs heterotrophs - Where it is that they get their energy from
- Chemorganotrophs, Chemolithotrophs & Phototrophs etc.
- think: teacher or doctor gets paycheque from that
Energy Classes of Microorganisms
Chemorganotrophs
• Energy from chemical reactions involving ORGANIC material
ex: C6H12O6
- 12 e-‘s go into ETC, allowing for the prod. of energy
- gonna have a lot of energy avail. if you break them down
ex: us
- still have to eat dinner to supply ourselves & not just sunlight b/c we LACK the photosynthetic machinery
Energy Classes of Microorganisms
Chemolithotrophs
• Energy from INORGANIC chemical reactions
ex: H2S (need to eat more of this, work more to get same energy yield)
Energy Classes of Microorganisms
Phototrophs
• Energy from light
____ will be chemical
Chemotrophs
chemorganotrophs, chemolithotrophs
How much energy is avail. in H2S as compared to C6H12O6?
NOT AS MUCH –> not a lot of e-‘s
What will the compensation be b/t an organism that uses glucose to supply itself b/c its a chemorganotroph & an organism that is chemolithotrophic & uses H2S to supply its energy?
WORK HARDER & MORE & EAT MORE of H2S to produce SAME AMOUNT of energy as glucose
Carbon Classes of Microorganisms
Microorganisms grouped with respect to carbon source:
- Heterotrophs
2. Autotrophs
Microorganisms grouped with respect to carbon source:
Heterotrophs
• Use ORGANIC carbon for building cell carbon and biomass
Microorganisms grouped with respect to carbon source:
Autotrophs
- Use CO2 to synthesize cell carbon
* a.k.a. primary producers
What about if an organism uses palmatic acid (C16 fatty acid) for C & energy. What is its classification?
-O-C=O-(CH2)14-CH3
chemorganoheterotrophic
heterotroph - b/c using organic C (a fatty acid to build all its sugar & nucleotides etc)
&
chemorganotroph - b/c using an organic chemical to supply its energy
What about if an organism uses light energy & organic chemicals. What is its classification?
PHOTOHETEROTROPH
What are we in term of our chemical & nutrient requirements?
Can we sustain ourselves on hydrogen sulfide?
CHEMOTROPHS
NO - so CHEMORGANOHETEROTROPHS
- b/c organic chemicals & we use organic forms to supply our C
- most relatively relevant bacteria fit there as well
