In this article we will discuss about:- 1. Introduction to Blue Green Algae 2. What are Cyanobacteria? 3. Mechanism of Action of Nitrogen Fixation 4. Heterocystsa 5. Symbiotic Cyanobacteria 6. BGA as a Biofertilizer 7. Governments Initiative 8. Future Prospects 9. Production Methods 10. Application.
- Introduction to Blue Green Algae
- What are Cyanobacteria?
- Mechanism of Action of Nitrogen Fixation by Cyanobacteria
- Heterocysts in BGA
- Symbiotic Cyanobacteria
- BGA as a Biofertilizer
- Governments Initiative for BGA Mass Production
- Future Prospects of BGA Fertilizer
- Production Methods of BGA
- Application of BGA
1. Introduction to Blue Green Algae:
The economic importance of blue green algae (BGA), also known as cyanobacteria primarily lies in their agronomic importance as Biofertilizers due to their N2-fixing ability that helps them to grow successfully in habitats with low or no combined nitrogen. They are naturally found in most paddy soils and improve the fertility and texture of the soil.
Japanese scientist Watanabe et al (1951) developed techniques for mass cultivation of blue-green algae to be used as bio fertilizer in paddy fields. Venkataraman (1961) coined the term ‘algalizatiori to denote the process of application of blue- green algal culture in field as biofertilizer.
He initiated algalization technology in India and demonstrated the way how this technology could be transferred to farmer level who hold small lands. Both free-living as well as symbiotic cyanobacteria have been harnessed in rice cultivation in India.
2. What are Cyanobacteria?
Cyanobacteria or blue green algae, are an ancient group of gram negative prokaryotes. They are among the most essential organisms on earth because of oxygen evolving and nitrogen fixing ability using sunlight as the sole energy source. They comprise of about 150 genera and 2,000 species, some of the predominant Nitrogen fixing genera are Anabaena, Nostoc, Aulosira, Calothrix, Tolypothrix, Aphanothece and Gloeotrichia. Although they mainly exist as free living organisms, symbiotic relationship with some plant and animal species has also been reported.
Cyanobacteria inhabit almost all the habitats on earth; from bare rock to soil and from water to air. The nitrogen fixing genera of cyanobacteria are either unicellular or filamentous. The latter can either be heterocystous or nonheterocystous.
The morphology of cyanobacteria varies from unicellular to filamentous or colonial forms. The colonies are often surrounded by a mucilaginous or gelatinous sheath, depending on environmental conditions. Nostoc, a filamentous cyanobacterium, may produce spherical colonies as much as 3 or 4 cm in diameter. Some of the filamentous cyanobacteria have three types of cells namely vegetative cells, climate-resistant akinetes and thick walled heterocysts.
3. Mechanism of Action of Nitrogen Fixation by Cyanobacteria:
Cyanobacteria have specialized nitrogen-fixing cells called heterocysts. Heterocysts are non-dividing cells which have lost the capacity of oxygenic photosynthesis and contain the enzyme nitrogenase, which helps to fix atmospheric nitrogen. Nitrogen fixing cyanobacteria have developed mechanisms to protect nitrogenase from inactivation by oxygen.
Heterocysts provide the ability to perform nitrogen fixation through the exclusion of atmospheric oxygen and oxygenic photosynthesis. In non- heterocystous filamentous and unicellular nitrogen-fixing cyanobacteria, nitrogenase and photosynthesis occur in the same cell. It has been suggested that in such organisms, nitrogen fixation is separated from oxygenic photosynthesis temporarily.
Nitrogen fixation by BGA are dependent on factors such as climatic, biotic, and physico-chemical properties of soil.
4. Heterocysts in BGA:
Heterocysts are specialized cells of the filament characterized or distinguished from others by their thick wall, polar nodule(s) and homogenous contents. Heterocysts are usually found in the members of Stigonematales and Nostocales (except Oscillatoriaceae). They may be either terminal (Gloeotrichia) in position or inter-calary (Nostoc) or the genus may have both terminal and intercalary (Anabaena desikacharyiensis).
Structure of Heterocyst:
The pale yellow, thick-walled specialized cells, larger than vegetative cells are called heterocysts. The wall of the heterocyst is differentiated into three regions, an outer fibrous, middle homogenous and inner lamellar layer.
It has pore(s) at the pole(s) where it remains attached with the vegetative cell. The pores are plugged with polar nodule(s) or polar granules. The wall of the heterocyst is thicker towards the Polar Regions. The inner content is dense and appears to be homogeneous. Ribosomes are less in number but other granular inclusions appear to be absent.
Due to absence of phycobilins and chlorophyll a, photosynthesis does not take place. The thylakoids are present and are tightly packed due to reduction of inter lamellar space and are present more towards the periphery than the central region.
Due to low concentration or absence of pigment, the mature heterocyst appears to be empty. The two lipids, glycolipid and acyl lipid present in the lamellae are not found in the vegetative cells.
Function of Heterocyst:
1. According to Fritsch (1951) during vegetative period it secretes certain substances which promote growth and cell division.
2. It is the site of N2 fixation.
3. Fay (1968) pointed out that in addition to N2 fixation it also helps in growth and development of the filament.
4. Steward (1970) suggested the following possible roles of heterocyst such as:
i. Involvement in N2 metabolism,
ii. Growth and differentiation,
iii. Control of sporulation, and
iv. Anti-quated reproduction unit.
Nitrogenase activity in Heterocyst:
In Cyanobacteria, heterocysts are involved in fixing nitrogen. These cells have thickened, multilayered cell walls that restrict the diffusion of oxygen. Due to high respiratory activity they maintain low intracellular oxygen concentration. Heterocysts also lack photosystem II and therefore do not evolve O2 during light reactions.
Oxygen supply is regulated to a large extent by an oxygen binding protein called leg hemoglobin present in legume nodules. As a result the environment in heterocyst is anaerobic.This is necessary for nitrogenase enzyme to function.
The Cyanobacterial nitrogenase is a tetrameric FeMo protein (the dinitrogenase) and a site for N2 reduction in the heterocyst. The other subunit is a dimeric Fe-protein (the dinitrogenase reductase), encoded by a highly conserved nifH gene. The Fe protein has a Fe4S4 complex where ATP is hydrolyzed, providing necessary electrons to the FeMo active site. Oxygen inhibition of the enzyme occurs in this subunit, where the molecule can interact with the Fe4S4 complex. Conversion of atmospheric Nitrogen to ammonia is a complex process.
5. Symbiotic Cyanobacteria:
Symbiotically competent cyanobacteria have some excellent features that make them particularly significant in any attempt to extend the list of N2-fixing symbioses to include plants of commercial interest, such as cereals.
1. Unlike rhizobia, most symbiotic cyanobacteria carry their own mechanism for nitrogenase protection.
2. Cyanobacteria have a vast host range, and are not restricted to roots only, but can form symbiotic association with various plant parts.
3. Plant cyanobionts have two major features in common:
(i) The ability to differentiate specialized nitrogen-fixing cells known as heterocyst and
(ii) Dispersal through short, motile filaments known as hormogonia which infect host plant by chemotaxis.
4. Cyanobacteria generally supply their hosts with fixed nitrogen, although they can also provide fixed carbon to non-photosynthetic hosts. The major plant hosts are Bryophytes, Cycads, the Angiosperm Gunnera, the water- fern Azolla, and fungi (to form lichens).
The Azolla-Anabaena azollae (blue-green alga) is a symbiotic association between a eukaryotic aquatic fern and a prokaryotic alga. Although both are photoautotrophs, they reside together in a unique mutually beneficial ‘relationship’. Azolla provides an enclosed environment for Anabaena within the fern’s aerial dorsal leaf lobes.
In return, Anabaena sequesters nitrogen directly from the atmosphere, which is required by Azolla for its growth. Azolla and Anabaena have never been apart for 70 million years. Within this giant period of time, the two partners have co-evolved numerous complementary behaviors that make them more resourceful. Azolla is able to form green mat over water and is readily decomposed to ammonia and produces large quantities of Biofertilizers.
Close examination of an Azolla leaf reveals that it consists of a thick, greenish (or reddish) dorsal (upper) lobe and a thinner, translucent ventral (lower) lobe immersed in water. It is the upper lobe that has an ovoid central cavity, the “living quarters” for filaments of Anabaena.
The thick-walled heterocysts often appear more transparent, and have distinctive “polar nodules” at each end of the cell. The “polar nodules” may be of the same composition as cyanophycin granules (co-polymer of arginine and aspartic acid). Cyanophycin granules occur in many cyanobacteria, and may serve as a nitrogen storage product.
Once the cyanobacterium has entered the host plant, a number of morphological, developmental, and physiological changes occur. The rate of cell division is reduced, ensuring that the cyanobiont does not outgrow the host.
Although Azolla can absorb nitrates from the water, it can also absorb ammonia secreted by Anabaena within the leaf cavities. Rice is the single most important source of food for people and Azolla plays a very important role in rice production.
For centuries Azolla and its nitrogen-fixing partner, Anabaena, have been used as “green manure” in China and other Asian countries to fertilize rice paddies and increase rice production. Republic of China has 3.2 million acres of rice paddies planted with Azolla. Rice can be grown year after year, several crops a year, with little or no decline in its productivity; hence no rotation of crops is necessary.
In addition to nitrogen fixation, Azolla also has a number of other uses. It grows very quickly in ponds and buckets, and makes an excellent fertilizer (green manure).
6. BGA as a Biofertilizer:
Blue green algae are popular biofertilizers because of their innumerable advantages to the agro-ecosystem. The algal biofertilizers are environment friendly, unlike chemical fertilizers that damage the environment. They are cheap alternatives as they require low cost inputs. Although they do not show immediate results but they show cumulative effect after 3-4 years.
The main advantage of BGA is biological nitrogen fixation, also it enriches soils with organic matter and reduces the C: N ratio. The other uses of BGA are enhanced solubilization of immobile phosphates and producing growth promoting substances in the soil. They improve the physical, chemical and biological nature of the soil and are responsible for long term soil fertility (figure 3.7). BGA has been reported to reduce the pH of soil and helps the soil to retain exchangeable calcium.
BGA as a biofertilizer in Rice Cultivation:
The term algalization was introduced by Prof. G.S. Venkataraman for use of BGA as a fertilizer. BGA can contribute about 20-30 kg N ha-1 along with organic matter to the soil, which is quite significant for the economically weak farmers incapable of investing in costly chemical nitrogen fertilizers.
Many Asian countries like India, China, Vietnam, etc., have been utilizing BGA in paddy cultivation as the alternative to nitrogen. It has been reported that Nitrogen availability, particularly in the rice fields, to plants is increased due to application of BGA.
The other reason for BGA becoming popular as bio fertilizer is the presence of growth requirement conditions which are easily available in India.
In India, a general predominance of BGA such as Anabaena, Nostoc and Calothrix were found to be widely distributed throughout rice growing tracts except in acidic soils of Kerala, Assam and parts of Tamil Nadu. Other forms like Cylindrosporum, Tolypothrix, Scytonema and Aulosira had localised distribution.
The distribution of soils harboring blue green algae in India varies from as low as 7 to as high as 80 percent in different states. For instance, Uttar Pradesh soils are rich in Aulosira, whereas Mastigocladae is found in Gujarat, Cylindrospermum occurs in Karnataka and Calothrix is commonly found in Punjab soils.
BGA occurred at densities from 1.0 x 10-2 to 8.0 x 10-6CFU/cm -2 and their abundance was correlated to pH and available ‘P’ content of soil. During recent years, quantitative studies showed consistent presence of BGA at high densities in soils under rice cultivation in countries like India, Malaysia, Philippines and Portugal.
7. Governments Initiative for BGA Mass Production:
The awareness of utilizing algal bio fertilizer to supplement the nitrogen requirement of rice crop was introduced as early as 1939. The potential of cyanobacteria in rice field was demonstrated by Singh (1961). However, the systematic approach was undertaken at IARI and an algal bio fertilizer technology was developed. According to this, BGA were allowed to grow with soil as carrier and then applied at l0kg/ha dry soil based inoculum in rice fields.
A new technology was developed with the financial assistance provided by Department of Biotechnology, in which BGA were grown in a formulated medium in glass/polyethylene house and soil was replaced with lighter carrier (straw). In 1972 at IARI, New Delhi the first ever production of Cyanobacterial Bio fertilizer in India was reported.
Since then, significant progress has been made, mainly in the technology of production, identifying efficient strains, newer techniques of inoculation for better establishment of inoculated organism in various soils and in demonstrating their beneficial effect on the crop yield in field trials on different crops. Presently, several technologies are available, such as indoor technology and production is in defined growth media.
Most commonly used species in bio fertilizer production are Anabaena variables, Nostoc muscorum, Aulosira fertilissima, and Tolypothrix tenuis.
In 1990, Department of Biotechnology, Government of India, New Delhi established four major centers in different paddy growing areas of the country for acceleration and extension of works on algal Biofertilizers. The programme was launched under Technology Development and Demonstration Projects on Cyanobacterial Biofertilizers’ in U.P. (Lucknow), Tamil Nadu (Madurai), West Bengal (Calcutta) and New Delhi.
The main objectives of the Programme were:
(i) To develop low cost indigenous technology for mass production of cyanobacteria,
(ii) To isolate regional specific fast growing and better N2 fixing strains,
(iii) To develop starter inoculum,
(iv) To demonstrate the farmers in field, and
(v) To study the benefits on both economy and ecology.
As a result of these studies cyanobacterial bio fertilizer was found very useful, especially for small and marginal farmers of the country with the view point of both economy and ecology.
The pure culture of BGA are transferred to culture flask having Fogg’s medium for growth and provided sufficient light. Now this algal culture is used as a starter culture to initiate the mass culture of BGA.
In general, there are four methods of algal production that have been reported viz,:
(a) Trough or tank method,
(b) Pit method,
(c) Field method and
(d) Nursery cum algal production method. The former two methods are essentially for individual farmers and latter two are for bulk production on a commercial scale
After harvesting, well dried BGA is packed. The bags are stored in cool dry place. These BGA can be preserved for 3 years without losing its efficiency. A 10 kg of BGA inoculant is recommended for one hectare of flooded rice. The dried BGA flakes are introduced to the field after 10 days implantation.
The application of BGA to the crops is called algalization. It increases the yield up to 34percent in the rice field.
Methods of Application of BGA Bio fertilizer:
1. One packet (500 g) of ready to use multani mitti based BGA bio fertilizer is mixed with 4 kg dried and sieved farm soil in rice growing area
2. The above BGA bio fertilizer is broadcasted to standing water in rice field 3 to 6 days after transplanting rice seedlings.
3. The field should be kept waterlogged for about 10-12 days after inoculation to allow good growth of BGA.
4. Avoid using chemical nitrogen fertilizer, if necessary add a very low dose and that too only after a gap of 3-4 days.
5. Apply BGA for at least four consecutive seasons to have cumulative effect.
8. Future Prospects of BGA Fertilizer:
In the present time, chemical fertilizers are more in practice for crop production which affected the soil and environment quality and the sustainability of the agricultural crop production systems. The adequate use of biofertilizers helps in maintaining soil quality and thus provides a low-cost approach to manage crop yield along with protecting the environment.
Traditional conservation based method with modern technology can reduce farmers’ dependence on chemical fertilizers and pesticides, as well as reduce the farming costs and environmental hazards.
1. Emphasis on integrated plant nutrient supply concept involving biofertilizers should be integrated with low doses of chemical fertilizers in the cropping system for better results.
2. Major research focus should be on the production of efficient and sustainable biofertilizers for crop plants,
3. There is need for research that highlights on selection of effective species of cyanobacteria according to climate situations.
4. There should be a quality control system for the production of inoculants and their application in the field.
5. Agronomic and economic evaluation of cyanobacterial strains for diverse crops is required.
6. Establishment of “Bio-fertilizer Act” and strict regulation for quality control of biofertilizers is required to make BGA and other fertilizers acceptable to farmers.