MICR2000 - Environmental Microbiology

Pure culture methods of measuring microbial diversity

culture dependent

• e.g., streaking

• good for growing things that use sugars

• difficult to replicate environmental conditions in lab

  • unknown pH, temp, salinity, partner requirements (microbial food webs

• can’t culture all types of microbes - not good representation of microbial diversity

Great Plate Count Anomaly

viable plate count and most-probable-number techniques underestimate true diversity of microorganisms (<1%) can be cultured in lab

Culture independent method of measuring microbial diversity

using DNA or RNA - typically 16S rRNA

phylogeny

• evolutionary history of microorganisms using gene sequences

• objective measurement of diversity

What is needed for a DNA marker gene for phylogeny?

large macromolecules (high information content)

universally distributed since start of evolution

functionally constant

slowly changing in sequence

• relative change constant across all microorganisms so we can compare

• not too slowly that we miss differences bc of reversions

examples

• ATP synthases (ATPases)

• DNA polymerases

• Ribosomal rRNAs

Importance of 16S rRNA gene

Meets requirements for evolutionary chronometer (good marker gene)

• e.g., not too many or too little bases

Part of SSU (small subunit) of prokaryote ribosomes

most phyla of bacteria and archaea are represented only by environmental 16SrRNA sequences as they couldn’t be cultivated

Gene sequencing steps/how to make a phylogenetic tree

Isolate DNA (of separate cell types to compare)

PCR amplification

Sequence PCR amplified genes (AGTC sequence)

Sequence analysis

• align rRNA gene sequences

• compare differences

Generate phylogenetic tree

• based on relatedness

• length of branches indicates number of differences

3 lineages of cells

3 domains

• Bacteria (prokaryotic)

• Archaea (prokaryotic)

• Eukarya (eukaryotic)

Which bacteria on the phylogenetic tree are closer to LUCA?

thermophiles - like hot environments, suggesting LUCA originated in thermal environments

Major Bacterial Phyla

Proteobacteria

• medically relevant such as e. coli, klebsiella

gram +ve bacteria

cyanobacteria

• produce oxygen

Chlamydia

• cause chlamydia

Planctomyces

• unusual cell shape

Deinococcus

• radiotolerant, find in radioactive environments

Green nonsulfur bacteria, Thermotoga, OP2, Aquifex

• thermophilic

• close to LUCA

Domain bacteria phenotypic diversity within groups/phyla

Many phyla phenotypically diverse, physiology and phylogeny not necessarily linked

Major Archaeal phyla

Euryarchaeota

• methanogens

  • produce methane

• extremophiles

Crenarchaeota

• most are extremophiles

• some live in marine, freshwater and soil systems

Microbes that grow at different temperatures

Psychrophiles

• grow optimally at <15 degrees, max temp for growth <20 degrees, min temp <0

psychrotolerant

• can grow at 0, optimal 20-40

mesophiles

• optimal temp 15-40 degrees, usually 39

thermophiles

• optimal growth 45-80

hypethermophiles

• optimal growth >80 degrees

Microbes that grow at different pH

acidophiles

• optimal growth low pH (<6)

neutrophiles

• optimal growth btwn pH 6-8

alkaliphiles

• optimal growth high pH (>8)

Microbes that grow in varying levels of salinity

Halophile

• optimal growth at 1-15% NaCl 

Extreme halophile

• optimal growth 15-30% NaCl

Halotolerant

• can tolerate some NaCl but grow best in its absence

Microbes that grow in different pressures

barophile

• thrives at high pressure

• most grow in darkness and highly UV sensitive 

obligate barophile

• can’t survive outside of high pressure

barotolerant

• able to survive at high pressure but can exist in less extreme environments

How have different extremophiles adapted to their environments?

Psychrophiles

• higher content unsaturated fatty acids to keep membrane fluid at low temp

Thermophiles

• bacteria

  • rich in saturated fatty acids

• archaea

  • lipid bilayer fused into monolayer, increasing rigidity

Barophiles

• higher proportion unsaturated fatty acids keeps membrane from gelling at high pressure

Halophiles

• higher proportion of acidic proteins with negative charge prevents salts coagulating proteins

Acidophiles

• proteins with higher isoelectric point to keep them stable at low pH (makes them +ve)

• simplified electron transport chain

Oxygen and microbial growth

Aerobes

• require oxygen to live (21% or higher)

Anaerobes

• do not require oxygen

• may be killed by oxygen

Facultative organisms

• can live with or w/o oxygen

• typically grow better in presence of oxygen

Aerotolerant anaerobes

• tolerate oxygen even though don’t use it

Microaerophiles

• use oxygen at low concs

Toxic oxygen species

How aerobes deal with toxic oxygen species

toxic oxygen species produced during metabolism are metabolised by catalase into something safe

obligate anaerobes are oxygen sensitive bc they do not contain catalase - cannot detoxify reactive oxygen species

Classes of microbes according to carbon source

autotrophs

• use co2 as carbon source (carbon fixation)

heterotrophs

• one or more organic molecules, feed on autotrophs or autotroph products

mixotrophs

• organic compounds or co2 fixing

Redox tower

represents range of possible reduction potentials in nature

top = greatest tendency to donate electrons

bottom = greatest tendency to accept electrons

difference in redox couple is released as energy

oxygen is at the bottom for electron acceptors, thus more energy released for microbes using oxygen

How is energy conserved (stored in different forms)

Energy released in redox is stored in ATP

long term storage = insoluble polymers like glycogen which can be oxidised to gain ATP

Electron carriers

Shuttle electrons and/or protons

NAD+/NADH, FAD+/FADH

Work done in stages down redox tower, electron carriers are part of the tower (intermediates)

What metabolism needs

Energy

• for anabolism - building compounds

• from catabolism - breaking down compounds

• sources

  • chemical (chemo-)

    • organic (-organo-)

    • inorganic (-litho-)

  • light (photo-)

Carbon source

• co2 (autotrophs)

• organic molecules (heterotrophs)

• mix (mixotrophs)

Other molecules - nitrogen, phosphorus, sulfur, magnesium, nickel, calcium

Thus naming is chemolithoautotroph

• chemolitho = energy from chemicals - specifically oxidation of inorganic compounds

• auto = carbon source - co2, fixes into organic compounds

Distinguish between chemoorganotrophs, chemolithotrophs and phototrophs

chemo- vs photo- = get energy from oxidation of chemicals vs light

organo vs litho (subcategories of chemotrophs) = oxidise organic compounds vs inorganic compounds to get energy

chemoorganotrophs - typically heterotrophs (use organic compounds as carbon source)

chemolithotrophs - typically aerobic respiration, most are autotrophs (carbon fixing)

Mechanisms for producing energy

Fermentation

• anaerobic process

• use organic molecule as both acceptor and donor - only chemoorganotrophs can perform

• Substrate level phosphorylation is anaerobic and doesn’t require electron transport chain

  • but reduces NAD+ to NADH

  • fermentation reoxidises NADH to NAD+, allowing this to continue

• net 2 ATP per glucose

Respiration - requires electron transport chain

• anaerobic

  • lower ATP yield than aerobic, still higher than fermentation

• aerobic

  • oxygen as terminal acceptor

  • 38 ATP yield

Fermentation process

Substrate level phosphorylation

Glycolysis steps (catabolism of sugars)

1. ATP added to glucose

2. split into 2× 3-carbon compounds

3. NAD+ reduced to form NADH

4. 2x compounds further oxidised to pyruvate 

5. pyruvate reduced to lactate or ethanol

• uses electrons from NADH, NADH is oxidised

• making NAD+, thus pathway can cycle

Respiration process - specifically electron transport chain

Electron transport chain + chemiosmosis

• electrons transferred across chain to terminal acceptor, protons from NADH or flavoproteins pumped across membrane

• carriers in cell membrane arranged in order of increasingly +ve reduction potential, ending in terminal e- acceptor

• proton gradient created, ATP synthase converts proton motive force to ATP

Electron Carriers

• NADH dehydrogenases

  • binds NADH, accepts 2 protons 2 electrons

  • protons pumped across cell membrane

• Quinones (Q)

  • non-protein containing molecules that participate in electron transport

• Flavoproteins and Ubiquinone (Coenzyme Q)

  • pumps protons across membrane

  • flavoproteins lose their protons which are pumped across membrane

• Cytochromes (cyt)

  • proteins containing heme groups

  • accept and donate single electron via iron atom in heme

  • terminal cytochrome donates electrons to terminal electron acceptor 

Major forms of anaerobic respiration

Nitrate (NO3-) reduction and denitrification (nitrate reduction all the way to N2)

• denitrification main biological source of nitrogen gas

• want to denitrify all the way to N2 (safe)

• Nitrate (NO3-) → nitrite (NO2-) → nitric oxide (NO) → nitrous oxide (N2O) → N2

Sulfur oxidation and reduction

Other metals e.g., manganese

Different types of chemolithotrophs

Use different inorganic compounds for oxidising, these are on different positions on the redox tower and generate different amounts of energy, meaning depending on substrate being oxidised chemolithotrophs grow fast or slow

Sulfur oxidisers

• many sulfur compounds oxidised

• sulfate final product

• elemental sulfur can be deposited in cells as granules

Nitrifying bacteria

• oxidise ammonia and nitrite

  • nitrification = ammonia to nitrite to nitrate

• only small energy yields so growth of bacteria very slow

• 2 groups of bacteria to fully oxidise ammonia intro nitrate

  • nitrosomonas

    • ammonia → nitrite

    • inhibited by nitrapyrin - farmers use to keep ammonia in soil longer

  • nitrobacter

    • nitrite → nitrate

  • commamox - can do whole process of nitrification by self

• importance of nitrification

  • nitrate = key nutrient for plants

  • nitrification key step in waste water treatment - ammonia produced during breakdown of organic matter

  • nitrate produced by nitrifiers removed by denitrification

iron oxidisers

• very small energy yields

• oxidise ferrous iron (Fe2+) (soluble) → ferric iron (Fe3+) (insoluble)

• Fe2+ is stable only at low pH, thus iron oxidisers are acidophiles

• some grow at neutral pH if in anaerobic environment

• a lot of iron is oxidised and put through a simple transport chain to keep process going and cytoplasm pH at 6

anoxic vs anaerobic

anoxic = no dissolved oxygen (O2) but can have bound oxygen (e.g., nitrate)

anaerobic = no oxygen at all (not even in bound forms)

Photosynthesis types

Oxygenic

• water → oxygen

• use light to generate ATP and NADPH

• photosystem II

  • light splits water into oxygen, pushes electrons to high energy, fall down transport chain to photosystem I

• photosystem I

  • electrons pushed up again, flows down chain to NADPH

• Z scheme (II to I, graph of reduction potential looks like Z)

• ATP produced by

  • non-cyclic photophosphorylation (II)

  • cyclic photophosphorylation (I)

    • electrons can flow back to photosystem II from fd to cyt bf

• chlorophyll is the photosynthetic pigment

Anoxygenic

• cyclic process

• reducing power for CO2 fixation comes from inorganic electron donor (ferrous iron, H2S, NO2) instead of water

  • oxygen not produced

• bacteriochlorophyll

Organisation of photosynthetic pigments in phototrophs

Chl/Bchl

• Not in chloroplasts like in plants

• In Reaction Centres (RC)

• Antenna pigments (LH) funnel light to reaction centres

• Chlorosomes function as massive antenna complexes, capturing low intensity light

Carotenoids

• accessory pigments always present

• energy absorbed transferred to RC

• primary role as photoprotective agents, preventing photo-oxidative damage from toxic oxygen species

Citric acid cycle

pathway through which pyruvate is completely oxidised into CO2

initial steps (glucose to pyruvate) same as glycolysis

Provides energetic advantage over fermentation

Metabolic features unique to prokaryotes

Anaerobic respiration

• e- acceptors other than oxygen

• denitrification

Chemolithotrophy

• inorganic energy sources (e.g., nitrification, hydrogen, iron and sulfur oxidisers)

• methanogenesis - generate methane from co2 or organic compounds

• mehtanotrophy - co2 from methane

• nitrogen fixation - nitrogen gas to ammonia

Carbon cycle - oxic and anoxic processes

CO2 to organic matter is done by autotrophs - chemolithoautotrophs and photoautotrophs, autotrophic methanogens

Organic matter to CO2 (decomposition of organic matter) is done by heterotrophs - chemoorganotrophs usually, or methanotrophs, heterotrophic methanogens

Oxic (oxygen present)

• CO2 to organic matter

  • oxygenic photosynthesis

• organic matter to CO2

  • aerobic respiration

  • anthropogenic activities

• methane to CO2

  • methanotrophy 

Anoxic (oxygen absent, but can be in compounds)

• CO2 to organic matter

  • anoxygenic photosynthesis

• organic matter to CO2

  • anaerobic respiration

  • fermentation

• CO2 to CH4 (autotrophic)

  • methanogenesis (CO2 + H2 → CH4)

• organic matter to CH4 (heterotrophic)

  • methanogenesis (ethanoic acid → CH4 + CO2)

Methanogenesis

Only carried out by archaea

Autotrophic or heterotrophic (using acetate)

Methanogens role in cow rumen microbiome

In cow rumen

• cellulose hydrolysed into sugars

• sugars fermented into volatile fatty acids by other bacteria, producing CO2, hydrogen

• perfect environment for methanogens (anoxic), they use this hydrogen which could inhibit other hydrolysis enzymes

Permafrost risks

biomass from plants trapped in permafrost

global warming = earlier thawing

ponds become anaerobic, microbes degrade carbohydrates to generate CO2, making an anoxic environment

methanogens convert CO2 and hydorgen into methane which goes into atmosphere, longer growing seasons, more biomass, +ve feedback loop

Nitrogen cycle key processes

Nitrification

• ammonia oxidation to nitrate

• nitrapyrin inhibits

denitrification

• nitrate reduced to nitrogen gas

nitrogen fixation

• reduction of N2 to ammonia, making nitrogen biologically usable

ammonification

• breakdown of proteins into ammonia

anammox

• anaerobic ammonium oxidation

• NH4+ + NO2- → N2 + 2H2O

• occurs in anammoxosome which protects cell from toxic intermediates (hydrazine)

• removes ammonia from marine environments, wastewater

Nitrogen cycle oxic and anoxic processes

Oxic

• nitrification

• ammonification

Anoxic

• denitrification

• anammox

• nitrogen fixation

• ammonification

Nitrogen fixation (process, symbiosis with roots, regulation and importance)

Process

• uses nitrogenase (composed of dinitrogenase and dinitrogenase reductase) in redox reactions

  • sensitive to oxygen

  • in aerobic cells nitrogenase protected from O2 by

    • high respiration retes to use oxygen

    • slime layers

    • compartmentalisation

    • oxygen scavenging by leghemoglobin

• reduce N2 to ammonia

• energy comes from fermentation, photosynthesis, respiration

• 16-24 ATP for 2 ammonia

Symbiosis with roots

• bacterial nodules in plant roots fix nitrogen plants can use

• plants provide carbon source (organic acids) used in citric acid cycle for energy

Regulation

• Nif genes

  • highly regulated bc energy intensive process

  • nitrogenase highly regulated

• N fixation blocked by

  • presence of oxygen

  • higher concs of ammonia, nitrate

  • certain amino acids

Importance

• fix nitrogen from gas in atmosphere (n sink) to make it biologically usable

Early Earth conditions and early metabolism

Anoxic, hot

early metabolism

• anaerobic and chemolithoautotrophic

  • obtained carbon by fixing co2 to biomass

• lithotrophs produced organic carbon, leading to evolution of organotrophs

Surface origin hypothesis and Miller-Urey Experiment

cellular organisms rose out of organic and inorganic primordial soup on surface

temp fluctuation, meteor impacts, dust storms argue against this

supported by Miller Urey Experiment

• passed electricity through simple organic compounds (similar conditions to early earth)

• formed organic carbon and amino acids

Subsurface origin hypothesis

life originated at hydrothermal springs on ocean floor, stable conditions

conditions for organic molecule generation favourable

• slightly acidic, iron rich water

• precipitates, geochemical processes to create organic compounds

stages of development for cellular life

• cell envelope from montmorillonite clay vesicles

• synthesis of phospholipid membrane vesicles

• proteins to catalyse reactions rather than spontaneous and metal catalysts

RNA world theory

• first self-replicating systems may have been RNA-based

• DNA more stable, eventually becomes genetic repository

3 part system developed

• DNA

• RNA

• Protein

Timeline of microbial evolution

Methanogenesis

First phototrophs (anoxygenic)

cyanobacterial lineages used water instead of H2S, generated O2

O2 could not accumulate until reacted with ferrous iron to produce ferric iron, producing banded iron formations

Bacteria and archaea begun to use oxygen (more energetically favourable)

oxic atmosphere - new higher energy metabolic pathways, ozone layer to protect from UV radiation

unicellular eukaryotes

multicellular eukaryotes

Banded iron formations

O2 could not accumulate until reacted with soluble ferrous iron in ocean to produce ferric iron (insoluble), shown by banded iron formations

Led to iron oxide formation

Consequence of O2 for evolution of life

formation of ozone layer

• barrier against UV radiation

• allowed life to develop above ocean surface