Introduction to Biogeochemical Cycles

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The following slides are provided by Dr.
Vincent OFlaherty.
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The Nitrogen Cycle

• Growth of all organisms depends on the
  availability of mineral nutrients
• Nitrogen required in large amounts as an
  essential component of proteins, nucleic acids
  and other cellular constituents
• Abundant supply of nitrogen in the atmosphere -
  nearly 79 in the form of N2 gas - main reservoir
  of N

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• However, N2 is unavailable for use by most
  organisms because there is a triple bond between
  the two nitrogen atoms, making the molecule
  almost inert
• Needs v. high energy or specialised enzyme
  complexes to break this bond
• Haber-Bosch industrial process fixes N2 to NH3 -
  1000C and 200 atm pressure
• Some special microbes can carry out the process
  under normal conditions

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• In order for nitrogen to be used for growth it
  must be "fixed" (combined) in the form of
  ammonium (NH4) or nitrate (NO3) ions
• This problem occurs because most plants can only
  take up nitrogen in two solid forms ammonium ion
  (NH4) and nitrate ion (NO3- )

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• Major reservoir of N is atmospheric N2, other
  major stores of nitrogen include rocks in the
  earths crust and organic matter in soil and the
  oceans
• Weathering of rocks releases these ions so slowly
  that it has negligible effect on the availability
  of fixed nitrogen
• So, nitrogen often the limiting factor for growth
  and biomass production in all environments where
  suitable climate and availability of water
  supports life

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Sources of N for plants and animals

• Most plants obtain the nitrogen they need as
  inorganic nitrate from the soil solution
• Ammonium is used less by plants for uptake
  because in large concentrations it is extremely
  toxic
• Animals receive the required nitrogen they need
  for metabolism, growth, and reproduction by the
  consumption of living or dead organic matter
  containing molecules composed partially of
  nitrogen.

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The Microbiology of the N-cycle

• Microorganisms have a central role in almost all
  aspects of nitrogen availability and thus for
  life support on earth
• N2 gas is cycled from the atmospheric form
  through a number of inorganic and organic forms
  back to N2 - bacteria are the major organisms
  involved in the N-cycle, often specific species
  are NB

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• Some bacteria can convert N2 into ammonia by the
  process termed nitrogen fixation these bacteria
  are either free-living or form symbiotic
  associations with plants or other organisms (e.g.
  termites)
• Other bacteria carry out transformations of
  ammonia to nitrate, and of nitrate to N2 or other
  nitrogen gases

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• Many bacteria and fungi degrade organic matter,
  releasing fixed nitrogen for reuse by other
  organisms.
• All these processes contribute to the nitrogen
  cycle.
• We shall deal first with the process of nitrogen
  fixation and the nitrogen-fixing organisms, then
  consider the microbial processes involved in the
  cycling of nitrogen in the biosphere

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Stages in the N-cycle

• The stages in the N-cycle can be summarised as
  follows
• N2 fixation
• Ammonification/mineralisatio
• Nitrification
• Denitrification

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Nitrogen Fixation

• N2 is an inert gas - must first be reduced to
  ammonia (nitrogen fixation),which can then be
  incorporated into organic molecules by microbes
• A relatively small amount of ammonia is produced
  by lightning. Some ammonia also is produced
  industrially by the Haber-Bosch process, using an
  iron-based catalyst, very high pressures and
  fairly high temperature

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• But the major conversion of N2 into ammonia, and
  thence into proteins etc, is achieved by
  microorganisms- biological N-fixation
• Total biological nitrogen fixation is estimated
  to be twice as much as the total nitrogen
  fixation by non-biological processes

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• Type of fixation N2 fixed (1012 g/year106
  metric tons/year)
• Non-biological
  
• Industrial 50
• Combustion 20
• Lightning 10
• Total 80
• Biological
• Agricultural land 90
• Forest and non-agricultural land 50
• Sea 35
• Total 175

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Mechanism of biological nitrogen fixation

• Biological N- fixation - 2 moles of ammonia
  produced from 1 mole of nitrogen gas, at the
  expense of 16 moles of ATP and a supply of
  electrons and protons (hydrogen ions)
• N2 8H 8e- 16 ATP 2NH3 H2 16ADP 16
  Pi
• Reaction is exclusive to prokaryotes using an
  enzyme complex - nitrogenase

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Nitrogenase

• Consists of two proteins - an iron protein and a
  molybdenum-iron protein
• Reactions occur while N2 is bound to the enzyme
  complex. Fe protein is first reduced by electrons
  donated by ferredoxin. Reduced Fe protein binds
  ATP and reduces the Mo-Fe protein, which donates
  electrons to N2, producing HNNH

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Mechanism of biological N2 fixation

• 2 further cycles of this process (each requiring
  electrons donated by ferredoxin) HNNH is reduced
  to H2N-NH2, and this in turn is reduced to 2NH3

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• The reduced ferredoxin which supplies es for
  this process is generated by photosynthesis,
  respiration or fermentation (lots of energy
  required ATPs)
• Very strong functional conservation between the
  nitrogenase proteins of all nitrogen-fixing
  bacteria

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• Can mix the Fe protein of one species is mixed
  with the Mo-Fe protein of another bacterium, even
  if the species are very distantly related in the
  lab - still work
• N-fixing organisms are all bacteria
• Some free-living, others live in intimate
  symbiotic associations with plants or other
  organisms (e.g. protozoa)

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Nitrogenase and oxygen

• Nitrogenase is highly sensitive to oxygen -
  inactivated if exposed to oxygen, reacts with the
  iron component of the proteins
• Major problem for aerobes - orgs have various
  methods to overcome the problem
• E.g. Azotobacter sp. have the highest known rate
  of respiratory metabolism of any organism, so
  might protect enzyme by maintaining a v. low
  level of oxygen in their cells

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• Azotobacter species also produce copious amounts
  of extracellular polysaccharide
• By maintaining water within the polysaccharide
  slime layer, these bacteria can limit the
  diffusion rate of oxygen to the cells
• In the symbiotic nitrogen-fixing organisms such
  as Rhizobium, the root nodules can contain
  oxygen-scavenging molecules such as leghaemoglobin

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Examples of nitrogen-fixing bacteria ( denotes a
photosynthetic bacterium)

• Free living
• Aerobic
• Azotobacter
• Beijerinckia
• Klebsiella (some)
• Cyanobacteria (some)
• Anaerobic
• Desulfovibrio
• Purple sulphur bacteria
• Purple non-sulphur bacteria
• Green sulphur bacteria

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• Symbiotic with plants
• Legumes
• Rhizobium
• Other plants
• Frankia
• Azospirillum
• Clostridium (some)
• Frankia

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Symbiotic nitrogen fixation

• 1. Legume symbioses
• Most NB examples of nitrogen-fixing symbioses are
  the root nodules of legumes (peas, beans, clover,
  etc.).
• Bacteria are Rhizobium species, but the root
  nodules of soybeans, chickpea and some other
  legumes are formed by small-celled rhizobia
  termed Bradyrhizobium

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• Bacteria "invade" the plant and cause the
  formation of a nodule by inducing localised
  proliferation of the plant host cell
• Chemicals called lectins act as signal molecules
  between Rhizobium and its plant host - v.
  specific
• Bacteria form an infection thread and
  eventually burst into the plant cells - cause
  cells to proliferate - form nodules

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• Bacteria always separated from the host cytoplasm
  by being enclosed in a membrane
• In nodules - plant tissues contain the
  oxygen-scavenging molecule - leghaemoglobin
• Function of this molecule is to reduce the amount
  of free O2, protects the N-fixing enzyme
  nitrogenase, which is irreversibly inactivated by
  oxygen

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• Bacteria are supplied with ATP (80), substrates
  and an excellent growth environment by the plant
  -carry out N-fixation
• Bacteria provide plant with fixed N - major
  advantage in nutrient poor soils

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Other symbiotic associations

• 2. Frankia form nitrogen-fixing root nodules
  (sometimes called actinorhizae) with several
  woody plants of different families, such as alder
• 3. Cyanobacteria often live as free-living
  organisms in pioneer habitats such as desert
  soils (see cyanobacteria) or as symbionts with
  lichens in other pioneer habitats

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The nitrogen cycle

• Diagram shows an overview of the nitrogen cycle
  in soil or aquatic environments
• At any time a large proportion of the total fixed
  nitrogen will be locked up in the biomass or in
  the dead remains of organisms

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• So, the only nitrogen available to support new
  growth will be that which is supplied by NITROGEN
  FIXATION from the atmosphere (pathway 6)
• or by the release of ammonium or simple organic
  nitrogen compounds through the decomposition of
  organic matter (pathway 2 (AMMONIFICATION/MINERALI
  SATION)

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• Other stages in this cycle are mediated by
  specialised groups of microorganisms -
  NITRIFICATION AND DENITRIFICATION

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Nitrification

• Nitrification - conversion of ammonium to nitrate
  (pathway 3-4)
• Brought about by the nitrifying bacteria,
  specialised to gain energy by oxidising ammonium,
  while using CO2 as their source of carbon to
  synthesise organic compounds (chemoautotrophs)
• The nitrifying bacteria are found in most soils
  and waters of moderate pH, but are not active in
  highly acidic soils

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• Found as mixed-species communities (consortia)
  because some - Nitrosomonas sp. - are
  specialised to convert ammonium to nitrite (NO2-)
  while others - Nitrobacter sp. - convert nitrite
  to nitrate (NO3-)
• Accumulation of nitrite inhibits Nitrosomonas, so
  depends on Nitrobacter to convert this to
  nitrate, and Nitrobacter depends on Nitrosomonas
  to generate nitrite
• Nitrate leaching from soil is a serious problem
  in Ireland

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Denitrification

• Denitrification - process in which nitrate is
  converted to gaseous compounds (nitric oxide,
  nitrous oxide and N2).
• Several types of bacteria perform this conversion
  when growing on organic matter in anaerobic
  conditions
• Use nitrate in place of oxygen as the terminal
  electron acceptor. This is termed anaerobic
  respiration and can be illustrated as follows

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• In aerobic respiration (as in humans), organic
  molecules are oxidised to obtain energy, while
  oxygen is reduced to water
• C6H12O6 6 O2 6 CO2 6 H2O energy
• In the absence of oxygen, any reducible substance
  such as nitrate (NO3-) could serve the same role
  and be reduced to nitrite, nitric oxide, nitrous
  oxide or N2

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• Conditions in which we find denitrifying
  organisms (1) a supply of oxidisable organic
  matter, and (2) absence of oxygen but
  availability of reducible nitrogen sources
• Common denitrifying bacteria include several sp.
  of Pseudomonas, Alkaligenes and Bacillus. Their
  activities result in substantial losses of N into
  the atmosphere, roughly balancing the amount of
  nitrogen fixation that occurs/year

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Microbial N-Fixation