micro test 2!

What are micro-organisms

Any organism that is invisible to the naked eye (

Viruses/phages (obligate intracellular parasites)

have a small genetic element (RNA) in which it replicates in the host. They rely on the host for energy, metabolic intermediates and protein synthesis.



 

Ultra small archaea and bacteria

Have such a small genome that it is too small to support life so will need a host to grow, as it lacks essential genes

‘mimivirus'

* Larger genome and diameter and has protein-translating genes, Nucleic material to proteins


* Not quite alive

CRISPR (Clustered regularly interspaced short palindromic repeats)

* It is the bacterial and archaeal immune system

(adaptable for gene modification, silencing + cheap)

* Target DNA from viruses if they have a viral immune system
* Used to modify DNA

Microbial skin flora and Malaria

Humans are either highly attractive or poorly attractive to mosquitoes.

If you are highly attractive to mosquitoes you have low population diversity of microbes on your skin but in high abundance.

Poorly attractive to mosquitoes you have a high diversity of microbes and a more even distribution (More microorganisms that could consume the metabolites out of your skin)

Gut community microbiology determines…

how you metabolize food

E.g. mice example

Vagus nerve

* Metabolites influence function


* GABA, Gamma aminobutyric acid
* Interesting interactions between

Ratios can determine how much GABA is present and thus can influence brain function due to the community composition

Planetary tera-forming

The world was highly reduced, basically no oxygen

No SO4, NO3, PO4, Fe3+ or bio essential metals

CU,S bound up in non-reactive metals

* Methanogenesis, fermentation

 

Photosynthesis: includes O2

Terminal electron acceptor available

Wider variety of metabolites/reactions available

Metals released

Viruses, viroid's and prions

Near life e.g. viruses and viroid's, undergo replication but they cant grow and don’t have a metabolism or maintain homeostasis

Prions- proteins that can undergo replication, undergo evolution via natural selection

a micro-organism needs to

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* Organisms maintain homeostasis
* composed of cells
* undergo metabolism,
* adapt to their environment e.g. swimming towards food
* respond to stimuli e.g. express a particular protein,
* undergo evolution and reproduce

Archaea

* Have ether-linked lipids, mostly antibiotic resistant, extreme conditions


* Similar to bacteria, shape, size, multiply by binary fission, may have flagella, no nucleus or organelles, circular chromosomes
* Similar to eukaryotes as they have no peptidoglycan in cell wall, not troubled by antibiotics
* Bacteria cell walls have peptidoglycan but archea dont

Gram stain=

classify bacteria by cell wall composition

Gram +ve bacteria= simpler walls with a large amount of peptidoglycan (purple stain)

Gram -ve bacteria= less peptidoglycan and an outer membrane (pink stain)

Process of staining gram +/- bacteria

* Add a chemical compound called crystal violet, gets to peptidoglycan and stains it, goes through the sugars, doesn’t go through to inner membrane
* Use a fixative- iodine, causes a reaction between Crystal violet and cell wall components fixes the Crystal Violet to the cell.
* Then add ethanol and wash the outside of the cells (sugars and the lipid membrane) Peptidoglycan remains
* Gram -ve = transparent, so need to add safranin, so they look pink
* Gram positive= purple (still appears purple as a strong stain)

saturated and non-saturated fats

Saturated with hydrogen

Unsaturated= double bond

High temp environments- organism will have more saturated fatty acids

Antibiotics target peptidoglycan

Gram -ve more resistant as it has less peptidoglycan 

Some bacteria/archaea have a outer capsule of polysaccharide

Prokaryotes lack complex compartmentalization

Don’t have ER, golgi apparatus



They have infoldings of the plasma membrane - increase SA for a greater metabolism

Naming

Genus name is italicized and capitalized

Species epithet e.g. *coli* needs to be italicized but not capitalized



Binomial name

*Escherichia coli*

Then goes to *E. coli*

The names are often meaningful

classification

Kingdom (domain)



Phylum



Class



Order



Family



Genus



Species

single vs plural

Bacterium vs bacteria

Archaeum vs archaea

Fungus vs fungi

Genus vs genera

Binary fission

1 cell splitting into 2

It is still growth as they don’t increase in biomass but they grow due to their being clones of themselves

Growth can also be increasing the volume of the cell

 

carbon sources

energy sources

electron sources

Auto- CO2

Hetero- organic molecules

 

Photo- light

Chemo- oxidation of inorganic compounds

 

Litho= inorganic

Organo = organic

Bacterial growth

= 2^n / 2^g (g= generations (n)

Lot of biomass very quickly

Doubling time

1/td

units = hr^-1

Batch growth (culture)

Enclosed- contained, limited by volume(space)

Set amount of nutrients

bacterial growth phases

lag phase: slow growth, getting used to the conditions, modifying metabolism

Exponential phase: maximum growth rate as not limited by anything e.g. space, nutrients

stationary phase: growth is starting to be inhibited and rate is slowing down. Not net change in microbial growth

decline phase: Growth rate < cell death, substrate limited, secondary metabolite build up (Some will generate antibiotics)

Diauxie

This is due to: Multiple substrates

* Pick what they are going to use first, to ramp up its metabolism to deal with a particular substrate

 

* Picks substrate it gets the most energy out of, so it can outcompete other cells for the energy
* 2nd substrate, cant grow as fast



2nd lag phase, as the presence of the 1st resource is actively repressing the expression of enzymes involved in the metabolism of the 2nd one = Catabolite repression

Requirements from microbial growth

Energy

* Predominately light
* Chemical energy



Carbon

* CO2
* Organic compounds, CH4



Liquid water, N,P, S, Na and other elements, Temperature, Space, pH, nutrients, Oxygen conc, Water activity

Enzyme denaturation

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* pH, temp, solubility, chemicals



Becomes linear and unfolds



Can return back to normal protein (only some)

Water activity and salinity

Water activity= ability to loose water (evapourates out)



High water activity= loose a lot of water and something that has a low water activity that will pick that water up

jam preservation

Jam has v low water activity, the microorganism will desiccate the microorganisms

salt conc.

Water leaves organism to balance out salt conc

Salt can impact how well a micro-organism will grow

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Pink lake= hyperhalophilic archaea = love salt

Maintain salt balance in high salt conc lakes

oxygen

Oxygen is toxic to some micro-organisms

Making superoxide's, peroxides that can break the cell walls

uses superoxide dismutase to convert oxide to o2g

Why do micro-organisms need controlling

Food-spoilage

20% of spoiled food post-harvest



Prevent disease

* Food borne disease
* Campylobacteriosis -> campylobacter (common in chicken guts)
* Nosocomial (hospital-acquired) infections
* Control micro-organism in hospitals can prevent secondary infections 
* Pneumonia -> Acinetobacter sp.



Prevent industrial and experimental contamination

* Industrial settings
* Bioreactors
* Alexander Flemming - eating orange in lab and noticed he got penicillin growing on the orange peel and noticed there was bacterial inhibition= discovery of antibiotics

Disinfection

**Disinfection**: destruction or removal of viable vegetative cells, they are growing



* Not bacterial endospores

**Sterilization**:

**Sterilization**: complete destruction of viable vegetative cells and bacterial endospores

Antisepsis:

**Antisepsis:** chemicals applied to bodily surfaced to destroy or inhibit vegetative pathogens



* Can be bactericidal or bacteriostatic
* Topical disinfection e.g. spraying a surface with ethanol or adding Dettol to a cut

Chemotherapy:

**Chemotherapy:** chemicals used internally to kill or inhibit growth of cells within host tissues

Sanitization:

**Sanitization:** microbial populations reduced to levels to be considered safe



* Reducing the number of microbial populations
* E.g. milk produce a production that will have micro-organisms but sanitized to a level that will the product will be safe  over the time you are likely to use it
* Treat it so pathogens aren't there

**Microbial death**:

the loss of membrane integrity and loss of electrical potential across the membrane

**Mechanical removal (filtration)** sterilization

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**Depth filters**: bonded fibrous or granular material (asbestos, diatomaceous earth(diatom shell))

larger gaps between where microorganisms can move but will eventually be intercepted over time and  absorbed diatomaceous earth.

 

Replies on electrostatic attraction and diffusion, interception of particles as they move through the filter

Good for large volumes air and gas

Not good for liquids, liquids will move through and wont be intercepted

**Mechanical removal (filtration)Laminar flow hood-**

Laminar flow hood-

 

Air comes in through the top through the big filter and blows clean air through

Environment where there is no micro-organisms.

 

HEPA filter= \[physical way of filtering micro-organisms from the air

Membrane filter (mechanical filter)

Membrane filter

* Very thin layer of material that has small holes in it, used for liquid with microorganisms, microorganisms trapped on one side and the liquid on others (0.1mm for cellulose acetate, 0.2 um for bacteria cells)
* Can vary in pore size depending on the micro-organism



Post-grad accidentally used too small of a filter and found micro-organisms that were extremely small, new phylum of bacteria

**Physical control methods**

Temperature

* Increase temp beyond the t op tmax get cell lysing and not being able to grow, too high all the cells will die

Moisture based temp by using an autoclave

**Moisture based temp by using an autoclave**



* Water in there, then crank handle up to increase pressure and get to 121 deg c and 15psi,. Will kill all organisms both vegetative and spores



All at different temps

Yeasts in vegetative state, 5min and 60 deg they will die

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Ensure all are killed at 15 psi and 121 C but not extremeophiles

physical method, dry heat e.g. oven

160 deg C for 2 hrs-  has to be for 2hrs as heat transfer is less efficient as it is drier heat is transferred a lot better through water

Moist heat kills micro-organisms quicker

 

Use Bunsen burner, way above 60deg

Pasteurization ( disinfection and sanitization)

* Heat it to about 60deg and will kill 99.99% of the pathogens/micro-organisms


* Going it enough so that it can have a longer shelf life e.g. UHT milk

Boiling liquids  (disinfection and sanitization)

* Will only kill vegetative cells (not spores)


* Microorganisms need to be at a certain conc. Or grow in certain conditions for you to get sick

**Ionizing** radiation (physical control)

Sterilization technique



* Damages DNA, lipids, proteins ( electrons, OH- radicals, H radicals
* Indiscriminate and moves through the system well
* Can kill you very quickly
* Radiation measured in grays, lethal dose for humans = 5 Gy

**Non-ionizing** radiation (physical control)

UV treatment = disinfection



220-300nm



UV light changes the DNA structure, get complexes between adjacent nucleotide



Get Thymine dimer, binding together on DNA strand causing a kink,  which means that the polymerase cant go along and read that dna as there is a kink

chemical control methods

Chemical agents via gases or liquids

e.g.

liquids

Phenols and alcohols- which denature proteins and dissolve membrane lipids (alcohols such as ethanol will sanitise)

Halogens- oxidation of cellular material

Aldehydes- inactivates nucleic acids and proteins

Gases e.g. Ethylene oxide which are very toxic

toothpaste and Triclosan

Most toothpaste/ personal care products will have Triclosan which is Anti-microbial, issue as micro-organisms can help break down micro-organisms, also going into waterways, brings about anti-microbial resistance

Inhibits enzymes used in fatty acid synthesis

Antibiotics, antimicrobial agents produced by micro-organisms

Variety of modes of action



Some impact peptidoglycan, ribosomes, lipid photosyntheise



Micro organism generate these to beat other microorganisms and compete with them



Then they have methods to deal with antibiotics/microbial

Anti-antibiotics

Generate pumps to pump out antibiotics fast



Compounds that will absorb the antibiotics in order to render them useless



Issue as the antibiotic resistance mechanism are sitting on plasmids and get transferred left and right to other micro-organisms



Need to finish the course of your antibiotics as you might start selecting for  micro-organisms with antibiotic avoiding capabilities.

**Biological control methods**

Viruses are used to control micro-organisms involved in food spoilage/food related illness

E.g. in cheese use phages that specifically tackle infections

Predator and viruses= antisepsisToxin= sterilization



*Bdellovibrio*= bacterial parasite (bdello= leech)

Feeds on biopolymers( proteins and nucleic acids) of their host. They are unable to replicate outside of the hosts cell

**Metabolism**

**s**um total of anabolic reaction and catabolic reaction

anabolism and catabolism

**Anabolism**: reaction that is taking something that is simple and generate something more complex, needs energy



**Catabolism**: Reaction that involves breaking down complex molecules to use the energy released for work

micro-organisms and energy usage

Micro-organisms take advantage of the 1st and 2nd laws of thermodynamics (Taking energy and converting it to something useful)



Micro-organisms are bags full of catalysts that efficiently harvest energy for growth and maintenance

how bacteria gets energy

Bacteria isnt getting any C from the substrates so uses some energy made to absorb CO2 as it needs carbon to grow.

Cells cant use heat energy to drive work



Cells cant capture and use all the energy from oxidation reactions at once

**Redox reactions**

Reactions where electrons are moved from a donor to an acceptor of electrons



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**Standard reduction potential (E0')**:

measure of the tendency of the donor of a half reaction to lose electrons

* The more -ve the E0', value the more likely the donor will donate an electrons

delta**E0' = E0 acceptor pair - E0 donor pair**

Not every reduction reaction is for energy generation (Dissimilative and **Assimilative reduction**)

**Dissimilative reduction**: reductive reactions of electron acceptors used for energy generation

**Assimilative reduction**: reductive reactions of electron acceptors used to acquire essential components ( C,N,S) for the synthesis of biomolecules

losing electrons in redox reactions.

**Electron is high energy and is going to low energy as it moves through the system**



During redox reaction

Every time it goes through a complex/system it decreases in energy

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doesn’t go from A to B in one reaction as they cant capture and use all the energy from oxidation reactions at once

Does steps down the redox tower in little bursts

* Donates to a NADH small amount of energy to pump the proton out


* Then goes through the quinone system
* Then transferred to cytochrome bc complex
* Transported to cytochrome C

Enzymes

Enzymes are the catalysts of the biological world

Allow you to go past the activation energy as they reduce the energy activation requirement

Exergonic/endergonic reactions

**Exergonic reaction:** reaction where energy is released and the reaction is considered spontaneous

 

**Endergonic reaction:** reaction that requires energy input to be driven and is considered non-spontaneous

free energy change

**Free energy change:** the amount of energy available to do useful work ( Delta G0) for microbes

If the free energy is -ve (energy of products smaller than substrates) = exergonic



If the free energy is +ve(energy of products is greater than substrates)= endergonic

free energy availability

If there is free energy available, there is enough energy to give you a -ve change in -deltaG0, then a micro-organism has found a way to use it and then grow

Energy conservation

A microbe aims to convert external energy sources as efficiently as possible to biomolecules (anabolism) or easily usable internal energy sources (catabolism)



Either



* High energy stores (glycogen, PHA's)
* Adenosine triphosphate (ATP)

They aim to do this with the minimum amount of energy loss. This energy conservation comes in the form of

1. Oxidative phosphorylation

* The oxidation of substrates to generate ATP

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2. Substrate-level phosphorylation

* The removal and direct transfer of PO3- from substrates to ADP (To make ATP)
* Energy released from getting the phosphate off generates ATP

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3. Electron bifurcation

* Coupling exergonic and endergonic electron transfer reaction to limit energy loss
* Simultaneously at the same time ender and exergonic

ATP and the energy currency of cells

* ATP is a cells energy storage system
* ATP is used to drive cellular endergonic reactions (anabolism)

How is the energy stored/used

* Free energy from catabolic reactions is used to generate ATP
* ATP is used to drive endergonic processes

How are electrons transferred between oxidants and reductants to allow for maximal energy conservation?

They use electron carriers( co-enzymes)



Accept energetic electrons move them to where they are needed then release them



Electron carriers



* Cells generate coenzymes that accept/donate electrons in reaction
* They carry electrons from one reaction center to the next
* The each have a different standard redox potentials.

e.g. Nicotinamide adenine dinucleotide phosphate \n (NADP+/NADPH -320 mV) \[same as NAD+/NADH\]

Each coenzyme

* Accepts electrons from more redox -ve donors and donates electrons to more redox positive acceptors
* Microbes maximize their ability to conserve energy derived from oxidation reduction reactions

Electron bifurcation

Putting electrons down here to less energetic states, but there is still excess energy which is used to boost the other electrons to a more energetic state, used for more complex processes/ processes that require more energy



Cells requiring more electronegative source an turbo-charge electrons for difficult/energetically demanding reactions can use electron bifurcation.

**Glycolysis**

Refers to all pathways used to break down glucose to a pyruvate.



* Uses substrate level phosphorylation- when adding on a phosphate then pulling it off again to generate ATP
* Anoxic- doesn’t need oxygen, or a terminal electron acceptor
* Takes glucose and makes a pyruvate
* Final products = Oxidized = multiple ATP, NADH, and 2 pyruvate

ASKS THIS QUESTION Does it have a terminal electron acceptor (something to accept the electron via the consumption of glucose) ?

Yes= TCA and respiration



No= fermentation (anerobic respiration)

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Yes, then goes to the CAC and ETC

**Citric acid cycle**

Pyruvate coming in and generate another NADH, carbons are donated, as being oxidized it is generating energized electron carriers and ATP (anabolic reactions to make compounds)m  = pyruvate oxidation



Products

3 CO2

4 NADH

FADH2

GTP (ATP)

**Oxidative phosphorylation**

As electrons are being passed, on the energy lost out , pumps protons out which helps form the proton gradient

 

Transfer electrons from NADH and FADH2 through the membrane protein complexes and ultimately to oxygen where they combine to form water. As electrons travel through the protein complexes in the chain, a gradient of hydrogen ions (protons) forms across the cytoplasmic membrane

 

Cells harness the energy of this proton gradient tot create 3 additional ATP molecules for every electron that travels across the chain

**Fermentation ( no terminal electron acceptor)**

Substrate level phosphorylation, to produce ATP

Need to generate more NAD+ which fermentation does



Ferments pyruvate

Generates 2 ATP

Needs to generate NAD+, (pyruvate is reduced by NADH)

**Products** lactic acid, Yeast ferments to alcohol, butanol, propanoic acid, H2

Cavities form due to fermentation

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Thick layer of plaque, no oxygen getting into the bottom, so generate lactic acid/organic acids, which goes into our teeth and form cavities

Not all micro-organisms use:

Glucose can be an energy or carbon source

Oxygen as a terminal electron acceptor

Can use; Sulfate, nitrate as a terminal electron acceptor instead of oxygen

Fixing carbon from carbon dioxide- non-organic

Metabolic diversity

Unlimited number of oxidation and reduction pairs



Need + standard redox potential



Have evolved to utilize any free energy to establish a niche

anaerobic respiration

The micro-organisms will find what their next best electron acceptor, closer to bottom of redox tower



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Reducing SO4 2-  -> S2-

                 Fe3+  -> Fe2+

                 NO3-  -> NO2-



Less energy as the difference in the redox tower isnt as big, so have less energy to pump protons across the gradient and then bring them back

Methanotrophs

Methane consumption by micro-organisms

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Methane donates electrons, Oxygen accepts them

deltaG= -ve, lots of energy, Can produce more ATP as making more energy (More energy per mole of methane)

Anaerobic methane-consuming archaea (ANME)

Mix of methanogenic (driving methanogenesis reaction in reverse) archaea and sulphate-reducing bacteria

Can sill use methane, archaea and bacteria working together

Produces sulfate

DeltaG = -ve, produces a whole lot less energy, all processes run a lot slower

red=Oxidises methane to co2

green=Reduces so4^2- to HS-

methanogenesis

Only archaea, generates energy from methane



CO2 is reduced by e-s donated from H2



V small amount of energy and produce very little energy with any substate

Metabolism Isn't straight forward, different orders of reactions, different substrates used in different environments/conditions

Hydrogen powered sulphite reduction

*Desulfovibrio vulgaris*

Hydrogen as an electron donor



Sulphite as electron acceptor

Difference between them = small, so very little amount of energy produced

How does *Desulfovibrio vulgaris* fit into central metabolism

* No glucose in the reaction


* Generates ATP, to fix carbon so it can grow
* Runs in reverse
* Not making co2, it is fixing co2- bringing it into the system
* Then passes it into glycolysis in reverse
* Anabolic, make more complex compounds to make nucleotides and fatty acids

Other autotrophs

ATP used for C fixation, ATP drives carbon fixation,CO2 made into complex compounds then runs through the system to



Yellow= central to all of live and they can run it in different ways depending on the substrate used

The fundamentals of central metabolism

energy metabolism, C-fixation remains the same it’s the sources that change

arsenic and methane

Arsenic in drinking water

Which is toxic, being mobilised by micro-organsims and can go into water ways



Increase in temperature causes permafrost to melt which causes methane to be released.

Good for methanotrophs, but if they consume all of it they release CO2 as a byproduct

why is the sulfur, iron and carbon cycles important

Sulphur is important for helping proteins fold

Can cause acid rain and thus the acidification of soils



Iron :Enzyme centers in proteins, haemoglobin

Corrosion, mining, climate change

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Carbon is needed for life

Photosynthesis, eutrophication, climate change

phototrophy

Phototrophy generates lots of compounds we need



Oxygenic phototrophy (cyanobacteria), H2O oxidised to , make oxygen

Anoxygenic phototrophy (purple and green bacteria),  other compound oxidised not oxygen e.g. So42- to H2S

Iron oxic and anoxic

OXIC

* Ferric iron = oxidizing agent
* Get orange colour
* aerobic microorganisms

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ANOXIC

* Fe3+ to Fe2+
* black colour
* anerobic micro-organisms
* Coupled with H2 or simple organics as energy donors

Fe and nanowires

Have nanowires Move electrons from iron rich to iron poor areas through this material

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Were able to oxidise hydrogen sulfide which requires oxygen in anoxic conditons.

* Via nanowires there was the transfer of oxygen via redox reactions between organisms

Sulphur oxic and anoxic

oxic

* Elemental sulphur reflect light to get that green-yellow colour
* oxidised

anoxic

* S0 to H2S

why cycle?

for energy flow

marine sediments and microbial fuel cells

Drilling programes with cores

Different colours due to, different microbial communities as you go deeper into the sediment

Based off oxygen conc, changing terminal electron acceptor e.g. nitrate



M.O on anode and oxidizes waste water, electrons stolen from micro-organisms to generate electricity

rumen and climate change

Cows

Evolved with methanogens in their system

Cow absorbs organic acids and uses it for energy, also generate H, for electron donor and CO2 for an electron acceptor to make methane

Cow doesn’t need methanogens there

Add a vaccine, wouldn’t produce methane as a waste product