Microorganisms & Ecosystems
Last revised: Thursday, April 10, 2003
Reading: Ch. 24 in text

Ecological Perspectives

• Ecology is the study of organisms and their environments. It asks different questions, and uses different methods, than the study of individual organisms and their internal machinery as we have been studying so far this semester.
• Example: A microbial ecologist might be interested in soil, or a pond. Typical questions might include:
  • what varieties of organisms are present?
  • How do they interact?
  • What happens to the population if environmental conditions change (e.g. hotter, drier, more acidic, etc.).
  • What changes occur if a new organism is introduced?
  • What happens if oil is spilled?
  • How long will it take for microbes to remove a pollutant?
• The answers to these questions are often very difficult to come by. Even the simplest sounding questions, e.g. "What organisms are present", is not easy to answer. If we rely on counting only those organisms that will grow on an agar plate in our laboratory conditions, we grossly underestimate many microbes that can be found by microscopic or other analytical techniques, but do not show up on a typical heterotrophic plate count.
• Microbes in nature have evolved stratagems both for competition and cooperation. For example, many microbes produce toxic compounds targeted at other microbes (antibiotics, bacteriocins, etc.). Some microbes are dependent on the metabolic activities of others; e.g., methanogens require H₂ produced by anaerobic fermentations. These organisms cannot be grown as pure culture in laboratory, since they require a mixed culture.
• Microbes adsorb to surfaces, creating biofilms. Biofilms consist of microbes and surrounding polysaccharide matrix. Much of what we know about microbes comes from studies of pure cultures in vitro. How this extrapolates to the behavior of complex mixed cultures growing in biofilms that are commonplace in nature we do not know.

PCR and Ribosomal RNA gene probes

• Technological advances can drive research. Good example is Polymerase Chain Reaction (PCR), a revolutionary technology.
• PCR is a technique to create large quantities of DNA from tiny samples (as little as one piece of DNA).
• Three steps in PCR process:
  1. Denaturation: heat the DNA to around 94^o C. This causes H-bonds holding A-T and G-C pairs to break, separation of two chains
  2. Primer Annealing: cool to 50^o C. Aadd primer (short piece of RNA 10-20 nucleotides long) to denatured DNA. Incubate to allow formation of primer-DNA base pairing.
  3. Polymerization: warm to 72^o C. Add DNA polymerase (isolated from thermophilic bacteria, so very heat-stable) plus dATP, dCTP, dGTP and dTTP.
• View animation of PCR cycling (requires Shockwave plug-in)
• Keep recycling these steps (all carried automatically inside closed box). Each time, double the amount of DNA: 1x 2x 4x 8x 16x 32x 64x .......... In 30 cycles, can get over 1 million molecules of product DNA!
• For 16S RNA studies, need to use primer that is found in every RNA molecule (universal primer), so probe will always find specific DNA site.
• Once sufficient DNA is produced, use DNA sequencing techniques to identify exact sequence.
• Application: use probe for small ribosomal RNA gene (in host DNA) to detect organisms in complex samples (soil, water, etc.). Any organism that has this gene can be detected.
• Astonishing result: enormous numbers of bacteria are present in soils, waters, that have never been isolated in lab. Less than 1% of microbial life forms have been cultivated!!! This "hidden diversity" means that microbes are undoubtedly doing all kinds of things that we don't know about.
• Methodological problems: the use of ribosomal RNA gene probes is not perfect. Errors can arise for various reasons:
1. DNA can only be probed if microbes are successfully lysed. Typically use a bead beater to break open cells.
2. Some rRNA genes are amplified better than others by PCR, so results can be skewed
3. Some substances ("humic" proteins in soils especially) inhibit PCR reaction, may be present in certain water or soil samples.
4. Not clear how much genetic variation in rRNA corresponds to different species.
5. rRNA results do not indicate anything about metabolism of microbes identified. Can't distinguish autotrophs from heterotrophs, for example.

Identifying Microorganisms in the Environment

• Ribosomal RNA gene probes have revolutionized the investigation of microbial communities. Many organisms that cannot be isolated can at least be identified as present. As we have discussed, this leads to estimates that less than 1% of microbes living in the environment have actually been cultivated.
  View diagram showing organisms identified by ribosomal gene probes. In this diagram, organisms identified by letter designations (OP3, OP11, etc.) have ribosomal RNA gene sequences that do not correspont to any known organisms. (From "Microbial Ecology and Diversity" by Norman R. Pace. ASM News Vol 65, Num 5, May 1999.)
• Microscopic methods: a variety of methods use light microscopy to detect microbes.
  1. classical stains such as phenol aniline blue; uses ordinary light microscope.
  2. fluorescent stains such as fluorescein isothiocyanate. These require a microscope illuminated with far-violet or ultraviolet light. Most microbes will bind the dyes, which adsorb the UV or violet light and emit visible light. The microscope contains a filter to remove the UV light from the path to the observer, so only the flourescence is seen. Fluorescent probes such as Mg-ANS (an 8-anilino-1-naphthalene sulfonic acid salt) can also be used. Their major advantage is that they can be applied to soil samples and immediately examined without removing excess, unreacted stain.
  
  
  Figure: Soil stained with Mg-ANS to show bacterial colonies and fungal mycelium (from University of Waterloo Biology 446 - Microbial Ecology course)
  
• Fluorescent in situ hybridization (FISH): use DNA probes with sequence that will complement sequence in rRNA. Attach fluorescent dye covalently to DNA probes (30-50 nucleotides, typicall). Treat cells with methanol to dissolve membranes, allow probe to reach inside of cell without completely destroying cell. Where probes bind, will see fluorescence under appropriate microscope. A new technique, very promising.

Organisms and Environments

• The study of ecology begins by accounting for the features of environments and the organisms found in them.
• What is an environment? It includes physical features (heat, wind); chemical features (pH, presence or absence of water, nutrients and minerals); and other organisms (which may be competitors, potential prey or predators, potential mates, etc.). These are called abiotic and biotic factors, respectively. Together, these factors comprise an ecosystem, which might be as small as a tiny pond or as large as the Coniferous forests that cover hundreds of thousands of square miles across North America, Europe, and Asia.
• If you are a "naked" microbe living on a beach, the most important factors in your survival may be the level of heat. If you are an E. coli bacterium living in the gut, heat is not a major concern; your survival probably depends more on your ability to compete successfully for the dregs of undigested food, and to withstand the various phages, colicins, and other biotic threats to your survival. And of course all this changes drastically the moment you are excreted into a sewage pipe.

Biogeochemical Cycling

• We saw earlier in the course that all organisms require the six elements CHNOPS in sizable quantities, and that these minerals may occur in different forms.
• In fact, depending on whether the local environment is aerobic or anaerobic, the exact chemical form of any of these elements may change quickly and drastically.
• Example: If a soil becomes water logged and anaerobic, sulfate will be converted into hydrogen sulfide and nitrate into nitrogen gas or ammonia (via anaerobic respiration). As water recedes and the soil becomes aerobic again, the ammonia is oxidized back to nitrate in stages.
• All of this (and many other processes) occur largely because of the activities of soil microbes. Without these microbes, life as we know it would not occur, because necessary elements would remain tied up in unusable chemical forms and gradually be removed by sedimentation.
• cycling and transfer of nutrients among all living organisms
  • biological and chemical processes involved
  • rocks/soil -- atmosphere -- water
• cycling of C, N, S, Fe, P, Mn occurs on a global scale
  • microorganisms crucial - profound effect on levels of different compounds available in environment
  • usually involves switching thru different redox states, and gaseous versus solid states
  • need to understand where substances produced, and where used (aerobic vs. anaerobic zones, etc)

Carbon Cycle

• The major cycling of C is from CO₂ to organic matter (by autotrophs) and from organic matter back to CO₂ (by respiration, burning of fossil fuels, etc.)
• In addition, however, there is a significant removal of CO₂ whenever hydrogen gas is produced anaerobically to form methane (anaerobic respiration by methanogens).
• This is not a trivial process; annual production of methane is about 180 x 10¹² grams of methane by biotic processes (belching from ruminants being the single most important) and an additional 130 x 10¹² grams by abiotic processes (mostly from incomplete combustion during burning of biomass).
• Interestingly, more methane is produced in freshwater and terrestrial environments than in saltwater, despite the fact that 2/3 of the earth's surface is oceanic. This is probably due to the high concentrations of sulfate in saltwater, which allows sulfate-reducing bacteria (which also need hydrogen gas in order to grow) to compete with methanogens for the available hydrogen.

Nitrogen Cycle

• This is the most complex cycle. Refer to lab exercise on the N cycle for an additional perspective.
• View animation of the Nitrogen Cycle
• Major reservoir = N₂ gas in atmosphere (80% of atmosphere) - most stable form
• Several key reactions carried out only by microorganisms
  1. Nitrogen fixation: N₂ NH₃
     • only carried out by certain bacterial genera: Rhizobium (root nodule symbionts), Azotobacter (free-living soil bacteria), others.
     • aerobic or anaerobic; ~ 60% on land; 40% in oceans
  2. Decomposition / ammonification: organic N NH₄⁺
     • carried out by chemoorganotrophs, both aerobes and anaerobes
     • some recycled into organic N in soil; some into atmosphere
     • excretion of extra nitrogen is often in some compound containing -NH₂ groups, which quickly form ammonia (NH₃) once they are liberated into the environment. That's why a baby's diaper smells like ammonia.
  3. Nitrification: NH₄⁺ nitrite, nitrate
     • mostly chemolithotrophs; some chemoorganotrophs in acidic areas
     • aerobic conditions; mostly well-drained soils at neutral pH
     • nitrate leached from soil by rainfall; water runoff from fertilized areas can become rich in nitrites, dangerous for animal health
     • inhibitors are sometimes added to fertilizers. E.g., nitrapyrin - inhibits NH₃ nitrite (1st step); decreases pollution of waterways
  4. Denitrification: nitrate N₂ or NH₃
     • chemoorganotrophs (anaerobic respiration)
  5. Assimilatory nitrate reduction: nitrate organic nitrogen
     • many organisms can carry this out
• Be careful to distinguish nitrification, denitrification as separate processes, and the conditions under which each occurs.
  
  

  Table: Nitrification vs Denitrification

  	Nitrification	Denitrification
  Aerobic status	Obligate Aerobes	Facultative
  Respiratory Classification	Aerobic Respiration	Anaerobic Respiration
  Respiratory electron acceptor	O2	NO3-, NO2-, others
  C-source	Autotroph	Heterotroph
  Energy source	Chemolithotroph	Chemoorganotroph
  Taxonomic group	Nitrosomonas, Nitrobacter	Variable group; many different genera
  Reversibility	Reversible	Not reversible

Sulfur Cycle

• Note that both assimilatory and dissimilatory sulfate reduction can occur, as we have seen with nitrate.
• View animation of the Sulfur Cycle
• Some major steps in the sulfur cycle include:
  1. Assimilative reduction of sulfate (SO₄⁼) into -SH groups in proteins.
  2. Release of -SH to form H₂S during excretion, decomposition, and desulfurylation.
  3. Oxidation of H₂S by chemolithotrophs to form sulfur (S^o) and sulfate (SO₄⁼)
  4. Dissimilative reduction of sulfate (SO₄⁼) by anaerobic respiration of sulfate-reducing bacteria.
     • carried out by mesophiles and hyperthermophiles
     • often same environment as methanogens, compete for available electron donors (e.g., H, acetate)
     • when sulfate present, sulfate reducers will win
     • only occurs when lots of organic C present (needed to generate electron donors)>
     • process often limited by organic C levels in marine sediments
     • pollution: sewage, garbage increase organic C, increase HS^-, H₂S production (both toxic to many organisms)
  5. Anaerobic oxidation of H₂S and S by anoxygenic phototrophic bacteria (purple and green bacteria)
• The sulfur cycle includes more steps than are shown here. Sulfur compounds undergo some interconversions due to chemical and geologic processes (not shown here). In addition, a number of organic sulfur compounds accumulate in significant amounts, especially in marine environments.
• For example, about 45 tons of dimethyl sulfide are produced annually by degradation of dimethylsulfonium propionate, a chemical produced by marine algae for osmoregulation. This is gradually broken down by a variety of biotic and abiotic mechanisms.

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