Nitrogen fixation in Algae, algal biofertilizers & algal blooms
Nitrogen fixation in Algae:-
A. Nitrogen fixing Blue Green Algae:- Both free-living and symbiotic blue-green are found to fix N2. Three groups of freeliving N2-fixing blue-green algae are recognized: heterocystous algae, nonheterocystous filamentous algae and unicellar algae.
1. Heterocystous blue-green algae:-
> These algae fix N2 aerobically and microaerobically.
> The most common N2-fixing species belong to the genera:
i. Anabaena
ii. Aulosira
iii. Calothrix
iv. Cylindrospermum
v. Nostoc
vi. Scytonema
vii. Tolypothrix
viii. Fischerella
ix. Mastigocladus
x. Stigonema.
> These algae usually differentiate vegetative cells into heterocysts only when grown in the absence of combined nitrogen.
> This observation led Fogg (1949) to suggest that heterocysts are the sites of N2 fixation.
> Nitrogenase is located in the heterocysts under aerobic growth conditions.
> Heterocysts are suitable sites for nitrogenase because oxygen evolving Photosystem II is absent in them.
2. Nonheterocystous filamentous blue-green algae:-
> Most of these algae fix N2 only under microaerobic conditions.
Exceptions:- Trichodesmium and Microcoleus are exceptions to this general observation, since they were found to fix N2 in aerobic conditions.
> The most common N2-fixing species belong to the genera:
i. Lyngbya
ii. Phormidium
iii. Plectonema
iv. Oscillatoria
v. Trichodesmium
3. Unicellular blue-green algae:- Gloeocapsa fix N2 in aerobic conditions while Synechococcus perform N2 fixation under anaerobic conditions.
4. Symbiotic blue-green algae:-
> Some heterocystous and unicellular blue-green algae develop in symbiosis.
> They are found in association with other organism:
i. With Diatoms:- Calothrix in Rhizosolenia.
ii. With Fungi:- Nostoc, Calothrix, Scytonema, Fischerella, and Gloeocapsa in lichens.
iii. With Bryophytes:- Nostoc in Anthoceros.
iv. With Ferns:- Anabaena in Azolla
v. With Gymnosperms:- Nostoc in Macrozamia
vi. With Angiosperms:- Nostoc in Gunnera.
> The importance of Azolla containing Anabaena azollae has been recognized by farmers of the Southeast Asian countries for centuries. Azolla is commonly used as a green manure to improve the nitrogen balance in rice fields.
> Contribution of blue-green algae of liverworts and lichens to the global nitrogen economy is probably of major importance.
> Cyanells:- Some organisms like Cyanophora paradoxa, Glaucocystis geitleri, Glaucocystis nostochinearum, Rhophalodia gibba and Glaucosphaera contain cell inclusions which resemble blue-green algae and these inclusions are referred to as cyanells. Of all these organisms, only Rhophalodia gibba was found to fix N2.
B. Biochemistry of Nitrogen fixation:-
1. Nitrogenase Enzyme:-
> Nitrogenase from bacteria and blue-green algae exhibit almost similar properties.
> It is a complex enzyme and consists of two iron-sulphur proteins, which, individually, have no detectable activity but together can catalyse the reduction of a variety of substrates (N2, N3-, N2O, HCN, CH3NC, CH3CN, C2H2, H3O+ and Cyclopropene).
> The larger protein (MoFe-protein or Dinitrogenase) of nitrogenase has high molecular mass (200000 - 270000 daltons), contains 18-36 iron, probably the same amount of acid-labile sulphur, one/two molybdenum atoms and consists of four subunits of two different types (structures).
> The smaller protein component (Fe-protein or Dinitrogenase reductase) has relatively low molecular mass (about 60000 daltons), contains a single cluster having four atoms each of iron and sulphur and consists of two subunits which are always identical.
> The MgATP2- activated Fe-protein accepts reducing equivalents from ferredoxin/flavodoxin and transfers to MoFe-protein with concomitant hydrolysis of ATP into ADP and inorganic phosphate.
> The Mo cofactor of MoFe-protein binds N2 and catalyses its reduction with reducing equivalents received from Fe-protein.
> For every molecule of N2 fixed, about 12-15 molecules of ATP are expended.
2. Requirement for reductant and energy:-
> To reduce N2 into ammonia, the enzyme has to be supplied with reducing equivalents and energy (ATP).
> The reducing equivalents for N2 fixation may be generated via:-
i. Both Photosystem I (PSI) and Photosystem II (PSII) from water
ii. PSI alone
iii. Dark reactions
> ATP may be provided by:
i. Cyclic and noncyclic photophosphorylation,
ii. Oxidative or substrate level phosphorylation.
> N2 fixation in blue-green algae is light dependent. It was found that C2H2 reduction takes place in far red light and in the presence of DCMU which inhibited CO2 fixation by blocking PSII.
> The dependence of N2 fixation on photosynthetic water photolysis is indirect. Since heterocysts lack the photosynthetic water-splitting reaction and RuBisCO, they depend on vegetative cells for the supply of carbohydrates.
> The light stimulated C2H2 reduction may be dependent on ATP generated in cyclic photosphosphorylation.
> In addition, light could also be necessary to supply the electrons to nitrogenase in a PSI dependent reaction. Alternatively, the electrons could be generated in the dark.
> Substrate level phosphorylation was demonstrated for Anabaena cylindrica but is believed to be of rather limited importance in providing ATP to nitrogenase. The immediate electron carrier to nitrogenase is ferredoxin.
> Blue-green algae contain a soluble, typical plant-type ferredoxin which mediate C2H2 reduction by nitrogenase.
> When Fe deficiency limits the biosynthesis of ferredoxin, several microorganisms form flavodoxin which takes over the function of supplementing electrons to nitrogenase. Since ferredoxins or flavodoxins are continuously transferring electrons to nitrogenase, they must be reduced by electron donors supplied from cell metabolism.
> The most important electron donor is probably NADPH. The formation of NADPH seems to be via hexosemonophosphate shunt where glucose-6-phosphate is degraded by glucose-6- phosphate dehydrogenase.
> The sources, other than glucose-6-phosphate, which may reduce ferredoxin are isocitrate, glycollate, malate and succinate.
> The transfer of electrons from all these donors to nitrogenase proceeds either in dark or in a PSI dependent reaction. PSI is definitely involved when H2 is the electron donor. In fact, H2 gave the highest activity among all electron donors in C2H2 reduction experiments with isolatd heterocysts.
3. Strategies to protect nitrogenase from damage by oxygen:- Since nitrogenase is highly sensitive to oxygen, N2-fixing blue-green algae have evolved different mechanisms to protect nitrogenase from oxygen.
a. Compartmentation:-
> Heterocysts function as compartments.
> These specialized cells are specially suited for activity of nitrogenase since they eliminate oxygen from them and are strongly reducing environments.
> They lack O2-evolving PSII.
> Further, respiratory oxygen consumption may remove the gas from nitrogenase site since the respiratory activity of heterocysts is higher than in vegetative cells.
> Hydrogen gas evolved during N2 fixation by nitrogenase also seem to protect the enzyme from oxygen since C2H2 reduction was found to be more stable against O2 addition in presence of hydrogen. > With regard to the O2 of air, a special function in binding O2 was suggested for four unique glycolipids found in laminated layer of the heterocyst envelope.
> Gloeothece possesses an elaborate system of internal membranes which may represent some intracellular protective compartment. It protects nitrogenase by a temporal separation of N2 fixation and photosynthesis, by building up a reserve of fixed nitrogen with little photosynthesis early in growth and later photosynthesizing without fixing N2.
b. Aggregation:-
> Clustering as an oxygen-restricting process has been reported in Trichodesmium.
> The filaments of Trichodesmium form bundles, on the outside of which photosynthesis occurs and within which N2 fixation takes place.
c. Mucilage:-
> Massive amount of mucilage produced by colony forming blue-green algae may arrest the diffusion of oxygen thereby protecting nitrogenase.
4. Nitrogenase and hydrogenase relationship:-
> H2 formation by blue-green algae takes place only under N2 fixing conditions.
> The formation of H2 is largely stimulated by light, but can also proceed in the dark, provided low levels of oxygen are present to allow respiration.
> These observations indicate that H2 must be generated by ATP-dependent reduction of H3O+ catalyzed by nitrogenase.
> H2 evolved by nitrogenase is not necessarily loss to blue-green algae. It can be utilized by two different pathways, both of which are catalyzed by hydrogenases.
> In the major pathway, H2 is consumed in an oxygen dependent reaction in respiratory chain and supplies organism with extra ATP.
> In the second pathway, H2 is utilized in a strictly light-requiring reaction. In this pathway, the transfer of electrons from H2 to the substrates has to proceed via PSI.
5. Regulation of nitrogense:-
> The regulation of nitrogenase biosynthesis has been extensively investigated in Klebsiella pneumoniae, where studies have established that glutamine synthetase is key control molecule.
> Glutamine synthetase turns into adenylylated form due to feed back inhibition by glutamine and stops nitrogenase synthesis.
> The de-adenylylated form of the enzyme is formed when this inhibition is removed and it is believed to promote nitrogense biosynthesis at the transcriptional level.
> However, in Anabaena, nitrogenase was not controlled by adenylylation.
> In Klebsiella, O2 not only destroys nitrogenase activity but also represses enzymes biosynthesis.
> Regulation by O2, light and other factors such as temperature, pH or nutrient supply may also occur in blue-green algae.
> Further, molybdenum deficiency severely decreased nitrogenase activity probably because of the production of inactive form of MoFe-protein under Mo starvation conditions.
Algal biofertilizers:-
Biofertilizers:-
> These are natural compounds that contain micro-organisms to enrich soil fertility to increase crop yield and plant growth.
> Microbial inoculants like bacteria, algae, and fungi can be used in biofertilizers.
> The biofertilizers having algae as an inoculant in them are known as algal biofertilizers.
> Biofertilizers can be used to fix nitrogen in the soil or they are also used to grow soil micro-flora to enhance soil health.
Algal biofertilizer:-
> BGA (blue-green algal) biofertilizers are used in fields to fix atmospheric nitrogen into the soil in organic form.
> BGA biofertilizers are not inhibited by the presence of any chemical fertilizers.
> Heterocyst is used to fix nitrogen into the soil.
Advantages of algal biofertilizers:-
> The algal biofertilizers can increase crop yield by 10-14%.
> Unlike chemical fertilizers they are eco-friendly.
> Low-cost input is required and thus is cheaper.
Benefits of BGA as a biofertilizer:-
> BGA is a biological nitrogen fixer, which concentrates the soil with organic matter and lowers the C: N ratio.
> Other uses of BGA are to improve the solubilization of immovable phosphates and to produce growth-promoting substances in soil.
> They improve the physical, chemical, and biological properties of the soil and contribute to long-term soil fertility.
> BGA has been reported to lower soil pH and help the soil retain exchangeable calcium.
> It has been reported that Nitrogen availability, particularly in the rice fields, to plants is increased due to the application of BGA.
> In India, BGAs such as Anabaena, Nostoc, and Carotrix are generally predominant and are widespread in rice-growing areas, with the exception of some acidic soils in Kerala, Assam, and Tamil Nadu. Other forms such as Cylindrosporum, Tolypothrix, Scytonema, and Aulosira had a local distribution.
> The prevalence of soils containing blue-green algae in India varies from 7% to 80% depending on the state.
Algal blooms:-
> An algal bloom is a rapid increase in the popullation of algae in an aquatic system.
> Algal blooms may occur in freshwater as well as marine environments.
> Result of an excess of nutrients (particularly p and n).
> Their growth - cause for other plants to die.
Harmful algal blooms (HAB):-
> A harmful algal blooms is an algal bloom that causes negative impacts to aquatic organisms via production of natural toxins, mechanical damage to aquatic organism.
> HABs are often associated with large-scale marine mortality events and have been associated with various types of shellfish poisonings and also fin fishes and other aquatic organisms .
> HABs in India:-
- Researchers have found toxic bloom has increased around 15 percent over the 12 years in indian seas.
- There was 80 harmful bloom recorded between the 1998-2010 in indian water.
> Worldwide Effects of HABs:-
- Light penetration levels in water decrease altering photosynthesis rate.
- Algal blooms may be harmful to seagrass and coral reef ecosystems and the connected food webs.
- Shellfish may accumulate algal toxins by feeding on the toxic phytoplankton resulting in fish kills, marine mammal distress, human illness and possible death.
> Reasons for Increase in HABs:-
- Global climate changes producing wider ranges for some species.
- Human contributions of increased nutrients and pollution in coastal waters and also fresh waters.
- Changes in local ecosystems that may allow exotic species to thrive if introduced.
- Upwelling.
- Formation of mud banks.
- Nutrients discharge on sea or fresh water through river.
- Eutrophication:- It is a process where by water bodies receive excessive amounts of nutrients, which results in excessive plant growth and formed algal bloom.
> Factors that can contribute to HABs:-
i. Excess nutrients (Eg.- phosphorus or nitrogen)
ii. Sunlight
iii. Low-water levels or low-flow conditions
iv. Calm water (low-wind conditions)
v. Warmer temperatures
> Examples of some HABs:-
i. Cyanobacteria (blue-green algae)
ii. Redtides:- Neurotoxic shellfish poisoning
iii. Ciguatera:- Gambierdiscus toxicus
iv. Alexandrium
v. Gymnodium
vi. Dynophysis
vii. Coolia monotis
viii. Prorocentrum lima.
Harmful effect of algal bloom:-
> Changes in levels of chemicals such as nitrogen and phosphorus from fertilizer, in the water.
> Algal blooms can deplete the oxygen and block the sunlight that other organisms need to live.
> Some can produce toxins that are harmful to the health of the environment- plants, animals, and people etc.
> Aquaculture industries.
Control method:-
> Biological:- Bacteria (Gymnodinium mikimotoi), Virus, Bivalves, Zooplankton etc.
> Chemical:- Copper sulfate, Alum etc.
> Physical