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CYANOBACTERIAL DINITROGEN FIXATION

Compiled by J.P. Keshri
The first suggestion that the Blue-green algae (Cyanobacteria) might fix
nitrogen came from the studies of Frank (1889) and Prantl (1899). The credit
however goes to P.K. De (1936) who most convincingly demonstrated the nitrogen
fixing ability of Anabaena inhabiting Indian rice fields. There is now positive
evidence for the nitrogen fixing ability of more than 100 strains or types of
Cyanobacteria. Although prokaryotes are photoautotroph and possess an oxygen-
evolving photosystem II very similar to that found in higher plants, their ability to fix
nitrogen photoautotrophically is based on two incompatible processes.
Nitrogen fixation is an inducible process, i.e., it is regulated by
environmental levels of ammonium or (to a lesser extent) nitrate. The process is one
of the most metabolically expensive processes in biology, requiring 16 ATP for each
molecule of N2 fixed. This energy demanding process therefore does not occur if
environmental levels of fixed nitrogen are sufficiently high, but triggered by low
levels.
Nitrogen fixation in Cyanobacteria is facilitated by the nitrogenase
complex which includes two major components: (1) The Mo-Fe-protein, which is a
tetramer formed of two types of proteins (encoded by nifD and nifK genes), several
iron-sulfur clusters, and two copies of a Mo, Fe, and S cofactor, which is active site
for nitrogen reduction, and (2) the Fe protein which contains two copies of the nifH
gene product along with an Fe-S cluster.
A second type of nitrogenase, which requires a vanadium cofactor, has
been identified in cyanobacteria and other nitrogen fixing bacteria. The vnf genes
encoding this second form are repressed by molybdenum. Magnesium and cobalt are
required for nitrogen fixation.
In the fully functional complex, electrons are first transferred from photosystem I
(via ferredoxin) to the Fe protein, then to the Mo-Fe protein. Three successive
transfers of electron pairs to Mo-bound N2 are required to produce two ammonia
molecules. GS (Glutamine Synthetase) is responsible for the assimilation of ammonia
produced by nitrogenase. Glutamine is exported to the vegetative cell (if the process
occurring in heterocysts) and with 2-oxoglutarate is converted by glutamine synthase
(GOGAT) to glutamate. In this way, fixed nitrogen becomes available for the

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production of other metabolites, although some of the glutamate must be transported

back to the heterocyst to complete the cycle.
The extreme lability of both nitrogenase proteins effectively prevents
nitrogen fixation and photosynthesis from operating in the same cell at the same time.
Therefore arrangements must be made to keep the two processes separate and yet
supply energy and reductant to nitrogenase and assimilate fixed nitrogen.
Consequently they have evolved special O2 protection mechanisms:

Heterocysts: The most common O2 protection mechanism one encounters is the

structural modification of the vegetative cell into heterocyst, which provides O2
protected environment for the nitrogenase. Heterocysts are large, thick walled
apparently empty cells growing in between pigmented cells on the algal filaments.
Differentiation of heterocyst involves structural and biochemical changes which help
in creating an anaerobic atmosphere for nitrogen fixation.
Many cyanobacteria produce thick mucilage, which may enclose heterocyst and
functions as a barrier to oxygen diffusion into cells. Heterocysts coated with mucilage
are able to fix nitrogen at significantly higher concentrations than were those lacking
mucilage. Bacterial cells associated with the surfaces of heterocyst commonly found
at the junctions between heterocyst and adjacent cells may consume oxygen thus
preventing its entry into heterocyst.
Structural modifications
1. Deposition of three layered envelope outside the cell wall. The outer layer is
fibrous, the middle one homogeneous and the inner one is laminated. The
laminated layer consists of glycolipids which reduce the rate of diffusion of
oxygen into the heterocysts. The middle and outer layer of heterocyst wall
consist of polysaccharides which may protect against degradation of the
glycolipid layer. This thick envelope not only limits the entry of oxygen to a
rate that is balanced by respiratory oxygen consumption, but allows sufficient
atmospheric nitrogen to enter in order to sustain nitrogen fixation.
2. Formation of a narrow junction between heterocysts and vegetative cells.
Polar nodules at these junctions prevent entry of external oxygen.
Microplasmodesmata at these points however permit metabolite exchange
required.
3. Mobilizations of granular inclusions and reserve products

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4. Disintegration and reformation of intracytoplasmic membrane systems into a

complex reticular pattern.
5. Degradation of several proteins and synthesis of new proteins. Carboxysomes
are degraded and a form of myoglobin called cyanoglobin that scavenge any
oxygen present preventing inhibition of nitrogenase is synthesized.
Biochemical modifications:
1. During heterocyst differentiation PS II components, Rubisco and light
harvesting chlorophyll binding proteins (LHCP) are degraded. While PSI
activity increases as compared to that in vegetative cells.
2. Heterocysts lack Hill reaction, light induced changes in fluorescence yield, Mn
complex and show reduced chlorophyll a florescence as compared to
vegetative cells. On the other hand heterocysts possess photosynthetic
electron transport (PET) of a PSI type action spectrum, nitrogenase activity,
light induced oxidation of P700 and Cyclic photophosphorylation. Thus,
although a functional PS II is absent, an active PSI is retained in heterocysts.
3. Heterocysts contain Fdx and Fdx – NADP+ oxidoreductase and an increased
amount of PSI to meet nitrogenase requirement.
4. During differentiation, phycobiliproteins (PBP) are mobilized into heterocysts
from vegetative cells. Subsequently, as enough nitrogen is fixed and becomes
available for protein synthesis, newly differentiated heterocysts synthesize
PBPs and become highly pigmented. The PBPs are qualitatively somewhat
different. They absorb light energy and transfer to chlorophylls in PSI. More
than the concentration of the PBP with photosystems, level of Mn, absence of
Chlorophyll binding proteins and cytb6 are factors leading to the lack of PS II
function in heterocysts.
Metabolic adaptations in heterocysts:
1. Carbon skeletons synthesized in vegetative cells are transported to provide
substrates for oxidative phosphorylation, reductive fermentation and to capture
newly fixed ammonia.
2. In heterocysts, very high rate of respiration is noticed. The high rate of
respiration helps heterocysts in two ways to maintain high rate of nitrogen
fixation (i) respiration helps in the break down of carbohydrate reserves
transported from vegetative cells via glycolytic pathway and oxidized through
hexose monophosphate (HMP) pathway for the production of energy and

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reductant for nitrogen fixation (ii) it reduces the oxygen tension in the
heterocysts. Enzymes and intermediate of HMP Such as glucose 6-phosphate
dehydrogenase, glycerol 3-phosphate, fructose 6-phosphate and 6-
phosphogluconate have been reported from heterocysts. Such intermediates
could enhance the oxygen uptake in isolated heterocysts. The enzymes of
Kreb’s cycle have also been reported from heterocysts.
3. Ability to utilize intracellular oxygen contributes to nitrogenase protection in
Cyanobacteria. Heterocystous Cyanobacteria have two types of hydrogenase
which differ in their location and functions. One is a classical type and is
found in soluble form in both heterocyst and vegetative cells while the other is
specific to heterocysts only. The latter type may enable the organism to utilize
molecular hydrogen for its growth, under nitrogen fixing conditions. This
class, called uptake hydrogenase, is involved in keeping a balance between the
reducing potential of heterocysts and helps in scavenging molecular oxygen to
protect nitrogenase. Hydrogen gas is produced by nitrogenase, which also
functions as a hydrogenase.
It is interesting that production of hydrogen gas in nitrogen fixation has
drawn interest as a renewable energy source as hydrogen fuel-cell technology
for motor vehicles becomes more of a practical reality (Schultz et al. 2004).
4. Uptake hydrogenase may not be sufficient on its own to take care of oxygen at
higher concentration. In Cyanobacteria, the reactive oxygen species
scavenging enzymes such as superoxide dismutase, catalase, ascorbate
peroxidase and glutathione reductase have been reported.

From the above account it is clear that heterocysts may be well treated as
‘anaerobic factories of nitrogen fixation under externally aerobic conditions’.

Nitrogen fixation in nonheterocytous Cyanobacteria:

Cyanobacteria exhibit multiple strategies to protect their nitrogenase from
oxygen. The problem is most severe in unicellular and in filamentous, non
heterocytous forms where photosynthesis and nitrogen fixation occur in the same cell
type. Salient features of the various oxygen protection mechanisms in these forms are
as follows.

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a) Temporal separation of Nitrogen fixation & photosynthesis: Temporal

separation of photosynthesis and nitrogen fixation was first proposed by Gallon
and his co-workers (1981) as a powerful mechanism to allow their co-existence
in nonheterocystous Cyanobacteria. They reported that in Gloeothece during
growth in alternate light: dark periods this cyanobacterium exhibits high rates of
photosynthesis in light while nitrogen fixation occurs only in dark. During
growth in light high levels of glycogen are accumulated and these are used up
later in subsequent dark phase for obtaining reductant and ATP. During
transition from light to dark nitrogenase proteins are synthesized afresh and the
same are degraded from dark to light phase of growth in each cycle. Moreover
this cyanobacterium develops an elaborate sheath around the cells during growth
under nitrogen starvation. The sheath may provide a barrier to oxygen diffusion
and thus protects nitrogenase. They also possess uptake hydrogenase and a
system analogous to ascorbate peroxidase and glutathione reductase which
afford metabolic protection to nitrogenase.
Nitrogenase in Oscillatoria can tolerate up to 0.15 atm of oxygen can
achieve a high rate of nitrogen fixation in dark. The protection mechanism in
this organism showed sensitivity to light, indicating photosynthetically evolved
oxygen may be more detrimental to nitrogenase than atmospheric oxygen and
the organism separates these two processes temporally to protect nitrogenase
from evolved oxygen. Another non-heterocystous filamentous cyanobacterium
Microcoleus chthonoplastes shows nitrogen fixing activity under aerobic
conditions, though low oxygen tension can induce nitrogenase activity. This
organism prefers light for nitrogen fixation over dark. Under completely aerobic
conditions, it fails to do nitrogen fixation in dark. Its ability to fix nitrogen in
dark at the expense of carbon reserves indicates that aerobic respiration can
provide reductant and ATP for nitrogen fixation but the preference for
diazotrophy in light suggests that an efficient mechanism for the protection of
nitrogen from photosynthetically evolved oxygen must be present in this
organism.
b) Enhanced continuous synthesis of Nitrogenase: In Cyanobacteria which
continually synthesize and degrade their nitrogenase daily, synthesis of
nitrogenase occurs at relatively high rates. Synthesizing and degrading
nitrogenase everyday is a terribly wasteful exercise but occurs in non-

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heterocystous forms since it is the only means of survival in nitrogen-deficient

environments, for such organisms.

c) Spatial separation of Nitrogen fixation & photosynthesis : A physical separation

of these processes in space occurs in several Cyanobacteria. Structural or
morphological differentiation under nitrogen fixing conditions has also been
reported in this regard in an aerobic non heterocystous cyanobacterium
Trichodesmium & Katagnemene. In the center of the colonized filaments of
Trichodesmium, certain highly pigmented cells (called diazocytes) devoid of both
CO2 fixing and O2 evolving activities are noticed. These cells function like
heterocysts cytologically nitrogen fixing cells have a denser thylakoid network
with fewer gas vacuoles and cyanophycin granules (Fredriksson and Bergmann
1997). However, isolate filaments of Trichodesmium do not show such cellular
differentiation or lack of nitrogenase and Rubisco activities in any trichome. It is
also a fact that, in nature, its nitrogen protection is via its ability to colonize
filaments in the form of bundles. Breaking these structures greatly inhibits
nitrogenase activity. The tropical seas where Trichodesmium & Katagnemene live
are relatively low in dissolved oxygen and this may assist the nitrogen fixing cells
in maintaining aerobic conditions in the protoplasm where nitrogenase is present
(Staal et al. 2003). A protective mechanism, involving reversible modification of
Fe protein of nitrogenase may be operative to combat high oxygen tension. The
co-existence of both forms of Fe-protein (i.e. 38 Kd and 40 Kd) in this organism
may be indicative of the presence of an active and an inactive form of nitrogenase
in cells. Similarly, lateral partitioning between photosynthesis and N2 fixation has
also been demonstrated in another nonheterocystous filamentous Cyanobacteria
Lyngbya aestuarii wherein photosynthesis is active in the intercalary regions
while nitrogen is fixed in the terminal cells. As a result such filaments show
contemporaneous photosynthesis as well as nitrogen fixation in day light while in
dark much of the filament is capable of nitrogen fixation.

Conclusive Remarks
It is well established Cyanobacteria are most important diazotrophs of
tropical and subtropical climate. They are the major contributors of the fertility of

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paddy fields. Moreover recent studies on bloom forming Cyanobacteria in the

tropical oceans suggest that they can generate as much as 85 x 109 Kg of fixed
nitrogen per year (Capone et al. 1997) approximately 35% of total nitrogen
fixation on earth. The filamentous cyanobacterium Trichodesmium has been
assumed to be predominant oceanic N2 fixing organism. In more recent studies
(Zehr et al. 2001) curiously unicellular Cyanobacteria of 3-10 m diameter like
Synechocystis and Cyanothece have shown significant nitrogenase activity. It has
been noticed that N2 fixation is higher at greater depths especially at 150M.
Moreover, N2 fixation rates are also higher during night. These unicellular
Cyanobacteria are heavily consumed by grazers as Trichodesmium is toxic to
some grazers; as a result the earlier data on nitrogen fixation were more
concentrated around Trichodesmium.
The basic steps of nitrogen fixation are more or less same in most organisms,
the modification occurred with respect to the protection of nitrogenase from
oxygen.