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Methods of increasing blue-green algal biomass
In this study the fate of five laboratory-grown heterocystous strains representing 75% of the inoculum was studied for 1 month in 1 m’ plots of five different soils.
Inocula are derived from a mix of strains isolated originally from rice-fields and grown in shallow trays with soil, phosphate and insecticide. If necessary, lime is added to adjust soil pH to 7.0-7.5. The blue-green algal mats which develop are allowed to dry and the dried flakes are stored in bags for use at 10 kg ha-' in farmers' fields. Algalization, the term widely used for the addition of such inocula. has received considerable publicity. Some reviews (Agarwal. 1979) have accepted the success of the method in raising grain yield as a well-established fact. Many studies have reported increased grain yield. grain nitragen content or straw nitrogen content (Venkataraman. 1981; Singh and Singh. 1987), with the effects of blue-green algae being equivalent to the addition of 20-30 kg ha-u nitrogen provided phosphorus fertilizer is added (Sharma and Gupta. 1983).
During the month following inoculation, the inoculated strains multiplied to some extent in all soils, but rarely dominated the indigenous blue-green algae and did so only when the growth of indigenous nitrogen-fixing species was poor or after population declines of indigenous species. The soils were dried at the end of the period and then resubmerged, together with neem, to control grazers. Two of the inoculated strains did not reappear, but one (Aulosira) developed an agronomically significant population on two soils. In field situations with a rich natural flora, it seems likely that indigenous strains will usually rapidly outgrow populations derived from the original laboratory isolates. Where farmers increase their inocula in the shallow trays, it is probable that strains present in the added local soil may outgrow the original laboratory isolates even before inocula are added to the fields. Efforts have been made to obtain strains with especially high nitrogen-fixing ability, so that these can be incorporated into the inocula. The approaches used have included screening of a range of strains obtained from enrichment cultures and attempts to obtain mutants in strains already in culture. The former approach has provided strains which are fast-growing in the laboratory (Antarikanonda and Lorenzen, 1982), but there is as yet no evidence that such strains have a particular advantage in the field. Although there are few quantitative data. many rice field blue-green algae probably often double every 1-3 days. It seems unlikely, therefore, that an introduced strain will survive long in competition with the natural flora. unless there is a simultaneous change in the environment which gives it a competitive advantage. It may prove useful to introduce fast-growing strains, if there is a sharp change in fertilizer practice. such as the use of high phosphorus, but no added source of nitrogen at a site which was not previously fertilized. , A further use for selected strains might be the addition of inocula resistant to pesticides. There are marked differences in the relative sensitivity of blue-green algae and weeds to widely used herbicides; the former are sometimes relatively insensitive. Overall, however, it is clear that the growth and activities of blue-green algae are affected adversely by some commonly used pesticides (Singh and Singh, 1983; Padhy, 1985b).
5 gm of top soil is taken from a paddy field and added in to a 100ml of fogg's medium in a flask. The flask is shaken well and incubated at room temperature. Now the illumination of 1500 lux is provided to the culture to increase the algal growth. Now the algal culture is transferred to 10ml of water in a tube with the help of a loop.Tube has to be shaken well so that algal filaments can be separated. The content is serially diluted. Every dilution drop is inoculated into Fogg's medium to establish algal growth. This process is executed in a petridish.
KH2PO2 - 0.2 g;
MgSO4.7H20 - 0.2 g;
CaCl2 - 0.1 g;
Na2MoO4 - 0.1 mg;
MgCl2 - 0.1 mg;
H3BO3 -0.1 mg;
CuSO4 - 0.1 mg;
ZnSO4 - 0.1 mg;
Fe-EDTA - 1.0 ml;
Distilled water - 100 ml; pH - 7
A drop from each culture is examined microscopically. If it is supposed to be a pure culture for a single species then only, it is used further. In case of more than one species, the sample is diluted till the isolation of a pure culture is attained.
The pure cultures of BGA are transferred to culture flasks having Fogg's medium for growth. To favour the growth sufficient light is provided. Now the algal cultures are used as starter cultures. This initiates the mass culture of BGA. It can be processed in four ways: Trough Method- In laboratory Zinc and Iron troughs are used. These are of 2K3 size and 22 cm height. The trough is filled with 10-12kg of soil and 200gm of super phosphate is spread on the soil. Now up to 5-15cms height water is poured. Calcium carbonate is added to adjust the pH around 7. Saw dust is provided to the soil. Now the starter culture is sprinkled over it. Trough is kept in sunlight where the BGA is developed very nicely. It is watered evereday. After sufficient response means nice growth of BGA the soil is allowed to dry. These dry flakes are collected and packed for algalization.
Under full sunlight shallow pits are maintained. To avoid the perlocation polythene sheets is lined inside the pit. The soil is filled in pit for 20cms and watered for 10 cm height. After maintaining the pH , carbofuran is added to the pit. Then saw dust is spread over the soil and is sprinkled with the starter culture. The pits are watered to favour the BGA growth. Thereafter, the soil is allowed to dry.
In an open field small plots of 40 sq mts are maintained. The plot is watered upto 15cms height and 20 kg superphosphate is added to it. After correcting the pH 240 gm carbofuran is spreaded in the field. Now the starter culture (5kg) is provided to the plot and is frequently watered. In 3-4 weeks BGA developed and then soil is allowed to dry. In this method, 30 kg of BGA inoculant can be harvested.
After harvesting well dried BGA is packed. The bags are stored in cool dry place. These BGA bags can be preserved for 3 years with out loosing its efficiency. A 10 kg of BGA inoculants 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 34 percent in rice fields.
The former authors used an algal material collected directly from the flask culture and blended after resuspension in distilled water, whereas the latter authors used an algal material dried at room temperature, comprising mainly vegetative cells in dormancy and akinetes, and therefore less susceptible to decomposition.
The basic method of mass production involves a mixture of nitrogen fixing cyanobacteria in shallow trays or polythene lined pits filled with water kept in open air, using clean, sieved farm soil as a carrier material. To each pit 10 kg soil and 250 g single super phosphate is added and water is filled upto a height of 12-15 cm. Starter culture, a mixture of Anabaena, Nostoc, Aulosira and Tolypothrix, is inoculated in each multiplication unit. Malathion ( 5-10 ml per tank) or carbofuran (3% granules, 20 g per tank) is also added to prevent insect breeding. In hot summer months, the cyanobacteria form a thick mat over the surface after 10-12 days of growth in open sun. The contents are allowed to dry and the dried flakes are collected, packed and used to inoculate rice fields. The basic advantage of this technology is that farmers after getting the soil based starter culture can produce the biofertilizer on their own with minimum additional inputs. An inoculum of 10-12 kg is considered sufficient to inoculate one hectare of paddy field 3-4 days after transplantation.
Unfortunately, the open-air algal biofertilizer production technology for production at farmers’ level is not popular among the farming community. The main limitations of this technology are:
- due to open air nature of production it can be produced for only a limited period in a year (3-4 months in summer; production has to be stopped during rainy and winter season),
- high level of contamination due to open type of production,
- slow production rate,
- low population density and hence need for heavy inoculum per hectare.
Therefore, efforts have also been made to improve the technology by developing new economically feasible protocols for production of quality inoculum so that these organisms can be practically exploited on a large scale. This is possible only if multiplication is carried out under controlled conditions. The production technology has been substantially improved with introduction of new and cheap carrier materials that support higher cyanobacterial load with longer shelf life, thus onsiderably reducing the quantity of inoculum per unit area. The basic changes the technology has undergone include, a) indoor production of algal biomass under controlled conditions; b) a suitable and cheap growth medium for faster growth of the organisms, and c) mixing with a suitable carrier material.
Nitrogen fixation measurements
N2-fixation by BGA has been most frequently studied using the acetylene reducing activity (ARA) method which may provide erroneous results (Lowendorf, 1982). ARA variations during the day and the growing cycle can be rapid and important; moreover ARA has a log-normal distribution (Roger et al., 1977). Therefore many replicates and very frequent measurements are needed to satisfactorily measure total ARA. However this tedious work will lead to an imprecise evaluation of the N2-fixing activity (NFA) because the conversion factor of acetylene- nitrogen is not constant and needs to be determined (Peterson and Burris, 1976). But ARA is a very convenient and reliable method for qualitative studies when the measurements are brief (David and Fay, 1977), when the problems of gas diffusion and greenhouse effects are minimized and when statistically valid methods are adopted (Roger and Kulasooriya, 1980). Few reliable estimations of ARA have been hitherto published. The number of measurements and replicates have been generally too low. Moreover, the importance of anaerobic nonheterocystous N2-fixing BGA was not appreciated until recently. Field measurements of nitrogenase activity were carried out under an aerobic gas phase only, therefore it is difficult to evaluate the N2-input due to N2-fixation by nonheterocystous BGA (Stewart, 1978). Reported data on BNF related to BGA varied from a few to 80 kg N/ha and averaged 27 kg/ha per crop (Roger and Kulasooriya, 1980).
Nitrogen-Fixing Capacity of The Alga
Any experiment designed to show nitrogen fixation by a given organism must be carried out with the following two points in mind:
(1) The organism must be in absolutely pure culture. Even if a contaminant is present which is known to be otherwise incapable of fixing nitrogen, the possibility cannot be precluded that it may fix nitrogen in the presence of the organism which is being examined.
(2) It must be absolutely certain that any increase in fixed nitrogen which takes place in a culture is due to the uptake of free nitrogen. Ideally, a manometric method, showing decrease in free nitrogen accompanying increase in fixed nitrogen should be used, but this is frequently inconvenient. A method showing increase in combined nitrogen is satisfactory provided that precautions are taken to exclude
the possibilities that combined nitrogen may be supplied from an unsuspected source, e.g. the atmosphere, or that the apparent increase may be due to some defect in the method of estimation. Since pure bacteria-free material of Anabaena shows vigorous growth in a medium free from combined nitrogen, it is probable that it is able to fix nitrogen. It is possible, however, that nitrogen is being absorbed in the form of ammonia or oxides of nitrogen from the atmosphere. In order to show that this was not so in the case of the algae studied by him, De (1939) carried out an experiment in which air was bubbled through sulphuric acid to remove ammonia, and potassium bicarbonate to remove oxides of nitrogen, before passing over the cultures. In the course of the present work it has been found that gas washing bottles of the type used by De are quite inefficient for this purpose, and, in the
absence of proof to the contrary, it must be assumed that all the combined nitrogen present in the air which passed over the cultures was not removed. De's(1939) contention that the increase in combined nitrogen observed in his cultures could not be due to absorption of ammonia or oxides of nitrogen from the atmosphere since uninoculated control flasks, exposed side by side with the cultures, did not show any
increase of nitrogen, cannot be regarded as valid since a growing alga would form a very much more efficient absorbing system for such substances than the medium alone. It is unlikely that the comparatively large increases in combined nitrogen found by De in his cultures were actually due to absorption of fixed nitrogen from the atmosphere, but this defect in his methods introduces an element of uncertainty which is undesirable in an investigation of this kind. An experiment similar to that of De, but using improved apparatus, has therefore been carried out with Anabaena cylindrica. Before passing over the cultures, air was purified from traces of combined nitrogen by passage through 1 % sodium bicarbonate solution and 25 % sulphuric acid (D), the wash bottles being of a type fitted with fritted glass bubblers. It was necessary to have the reagent with the lower vapour pressure (the sulphuric acid) nearer to the culture vessels since otherwise the cotton wool filter E became wet and allowed the cultures to become contaminated. The four culture flasks (F, G, etc.), one of which was left uninoculated as a control, were connected in series in the culture chamber. This part of the apparatus was sterilized and assembled under aseptic conditions and the air passing through it was sterilized by the sterile cotton wool filter E. A second sterile filter (H) and wash bottle containing sulphuric acid (J) were included respectively to prevent contamination and to absorb ammonia in the event of any sucking back. A, C and / were safety flasks. All connexions were sealed with paraffin wax. Air was drawn slowly (approximately 3 1. per hour) and continuously through the apparatus by means of a filter pump.
The efficiency of the gas-washing system was tested as follows. In each of two culture flasks was placed 100 ml. of iV/50 sulphuric acid (to absorb ammonia) and in each of two others 100 ml. of iV/50 sodium hydroxide (to absorb oxides of nitrogen). Air was drawn through the wash bottles and then through these flasks for a period of 10 days, the rate of flow being somewhat more rapid than that employed for aerating cultures. At the end of the experimental period the contents of the flasks were found to show no increase in combined nitrogen compared with controls analysed at the beginning of the experiment. This demonstrates that ammonia and oxides of nitrogen were effectively removed from the air passing over the cultures.
The cultures were inoculated from a pure bacteria-free stock culture. In order to ensure that the cultures had not become contaminated during the course of the experiment a small amount of material from each of the flasks was examined bacteriologically at the end of the experimental period by the same means as those used for the examination of stock cultures. No contamination by fungi or bacteria
The results of micro-Kjeldahl analyses of the cultures are given in Table 1. This table shows clearly that free nitrogen has been fixed by the alga. The small amount of nitrogen found in the control flask, the medium in which was completely free from combined nitrogen at the beginning of the experiment, may perhaps be due to ammonia derived from the culture flasks. It is to be noted that a large proportion of the nitrogen fixed appears in a soluble form in the medium. The nature of this excreted nitrogen is being investigated.
Table i. Nitrogen fixation by Anabaena in aerated culture after 50 days. Alga showing no sign of senescence. 100 ml. nitrogen-free medium per flask. Nitrogen in mg.
Mass cultivation of cyanobacterial biofertilizers
For outdoor mass cultivation of algal biofertilizers, the regional specific strains should be used. However, many germplasm collection laboratories have been established by the D.B.T. in different parts of the country for the development of starter inoculum. Mixture of 5 or 6 regional acclimatized strains of algae, e.g. species of Anabaena, Aulosira, Nostoc, Tolypothrix are generally used for starter inoculum. The following four methods are used for mass cultivation : (i) cemented tank method., (ii) shallow metal troughs method, (iii) polythene lined pit method, and (iv) field method. The polythene lined pit method is most suitable for small and marginal farmers to prepared algal biofertilizer. In this method, small pits are prepared in field and lined with thick polythene sheets. Mass cultivation of cyanobacteria is done by using any of the four methods under the following steps:
Prepare the cemented tanks, shallow trays of iron sheets or polythene lined pits in an open area. Width of tanks or pits should not be more than 1.5 m. This will facilitate the proper handling of culture.
Transfer 2 -3 Kg soil (collected from open place for lm 2 area of the tank) and add 100 g of superphosphate. Water the pit to about 10 cm height. Mix lime to adjust the pH 7. Add 2 ml of insecticide e.g. malathion to protect the culture from mosquitoes. Mix well and allow to settle down soil particles.
When water becomes clear, sprinkle 100 g of starter inoculum on the surface of water.
When temperature remains between 35-40° during summer, optimum growth of cyanobacteria is achieved. Always maintain the water level to about 10 cm during this period,
After drying, the algal mat will get separated from the soil and forms flakes. During summer about 1 kg pure algal mat per m2 area is produced. These are collected, powdered, kept in sealed polythene bags and supplied to the farmers.
The algal flakes can be used as starter inoculum if the same process is repeated.
Pot culture. The rice seeds were soaked in water for 20 days. Then 5 seedlings with the height of 2 cm were transferred to pots. One week before and one week after transferring the seedlings, 1 g of mixed wet algal inoculum was added to the soil. After three weeks, height of plant, roots length, fresh and dry weight were measured as per method suggested by Meloni, et.al.,(2004). Moisture (Hayes 1981), bulk density, particle density and porosity of soil (Blake and Hartage 1986) were also recorded.