inorganic phosphatic compounds soluble (Bardiya and Gaur, 1972; Wani et al., ... Gaur (1988) in his recent review on phosphate solubilizing microorganisms.
J. Biosci., Vol. 14, Number 3, September 1989, pp. 203-208. © Printed in India,
Extracellular phosphate solubilization by the cyanobacterium Anabaena ARM310 R. NATESAN and S. SHANMUGASUNDARAM* Department of Microbiology, School of Biological Sciences, Madurai Kamaraj University, Madurai 625 021, India MS received 21 April 1989 Abstract. Endogenous polyphosphate depleted Anabaena ARM310, solubilized extracellular tricalcium phosphate through increased phosphatase activity. Keywords. Extracellular phosphate solubilization; involvement of phosphatases.
Introduction Solubilization of fixed soil phosphates by microorganisms for greater assimilation by plants is of practical importance. Different groups of phosphate solubilizing microorganisms, particularly bacteria and fungi, have been reported to render inorganic phosphatic compounds soluble (Bardiya and Gaur, 1972; Wani et al., 1980). Gaur (1988) in his recent review on phosphate solubilizing microorganisms has implicated the organic acids produced by fungi and bacteria in phosphate solubilization. Cyanobacteria are increasingly used as bio-fertilizers for rice (Venkataraman, 1972). An efficient nitrogen fixing and phosphate solubilizing strain of cyanobacterium will be very useful for the cultivation of rice where bound phosphates remain unavailable. In the present investigation, the ability of Anabaena ARM310, a heterocystous nitrogen fixing cyanobacterium, to solubilize extracellular insoluble tricalcium phosphate using phosphatases was studied. Materials and methods Cyanobacterial strain Anabaena ARM310 was obtained from the Division of Microbiology, Indian Agricultural Research Institute, New Delhi. Culture medium and growth conditions Anabaena ARM310 was maintained in BG11 medium (Rippka et al., 1979). The pH of the medium was adjusted to 7·2 using 0·1 Ν NaOH before autoclaving. Cultures were maintained as batch cultures at 26±1°C with a light intensity of 1500–2000 lux, and grown in a light/dark cycle of 12/12 h. *To whom all correspondence should be addressed.
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Phosphate starvation The filaments of Anabaena ARM310 were subcultured in BG11 medium containing combined nitrogen but without dipotassium hydrogen phosphate. However, potassium chloride was added to maintain the availability of potassium in the medium. Twenty to twenty five day old cultures were used as inocula for experiments. Insoluble phosphate used in the experiments Tricalcium phosphate [Ca3(PO4)2], was added at a concentration of 1 mg/ml (w/v) of medium. Growth Pre-weighed phosphate starved cells were inoculated and subjected to the following treatments. (A) CN+ SP– –BG11 medium containing combined nitrogen but without dipotassium hydrogen phosphate. (B) CN+ SP+ –BG11 medium containing both combined nitrogen and dipotassium hydrogen phosphate. (C) CN+ ISP+ –BG11 medium containing both combined nitrogen and insoluble tricalcium phosphate. (D) CN– SP– –BG11 medium without combined nitrogen and dipotassium hydrogen phosphate. (E) CN– SP+ –BG11 medium without combined nitrogen but with dipotassium hydrogen phosphate. (F) CN– ISP+ –BG11 medium without combined nitrogen but with insoluble tricalcium phosphate. All the treatments were carried out in duplicates. The end point growth was recorded after 25 days of culture in all the treatments. Estimation of chlorophyll a Chlorophyll a was estimated by the method of Mackiney (1941). Harvested cells from each treatment were suspended in 1 ml of distilled water. Then 4 ml of methanol was added and the tubes were incubated in a waterbath at 60°C for 30 min. The tubes were then centrifuged for 10 min and the supernatant was used to read the absorbance at 663 nm. pH estimation The pH of the culture filtrates was measured once in every 5 days. The pH of the uninoculated media served as control.
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Phosphate solubilization and assay of phosphatase Phosphate solubilization was studied in terms of extracellular phosphatase activity in the culture filtrates. The activity of phosphatase was assayed on 10th, 15th and 20th day of growth. Alkaline phosphatase activity in the culture filtrates was assayed by suitably modifying the procedure of Freeland (1985). The alkaline phosphatase assay mixture contained 1·5 ml of 0·4 Μ Tris-HCl buffer (pH 8·5), 0·1 ml of 16·6 mM Phenolphthalein diphosphate and 1·4 ml of culture filtrate from the inoculated flasks, in a total volume of 3 ml. Tubes containing the assay mixture were kept for 30 min in dark at room temperature. After 30 min, the reaction was terminated by adding 0·1 ml of 1 Μ dipotassium hydrogen phosphate. The absorbance of the supernatant, which turned pink due to the release of Phenolphthalein, was measured at 540 nm. Uninoculated culture medium in a total volume of 3 ml of the assay mixture was used as blank. The amount of Phenolphthalein released was measured from the Phenolphthalein standard graph. Protein in the culture filtrate was estimated following the method of Lowry et al. (1951), using bovine serum albumin as the standard. The alkalaine phosphatase activity is expressed as µg of Phenolphthalein liberated per µg protein per min. Acetylene reduction assay Phosphate starved cells of Anabaena ARM310 were inoculated in flasks containing the treatments Ε and F. Nitrogenase activity was determined following the acetylene reduction technique of Stewart et al. (1968). Ten and fifteen day old algal samples were taken in 1 ml of medium in serum bottles (6 ml capacity) separately. The bottles were closed with a subaseal rubber stopper. After 6 h of stabilization in the medium, 1 ml of pure acetylene was injected into each bottle. The algal samples in serum bottles were incubated again for 6h under 1500 lux light intensity. The reaction was terminated by injecting 0·2 ml of 20% trichloroacetic acid. One ml of gas sample from each serum bottle was analysed using 5830 A Hewlett Packard microprocessor attached gas Chromatograph fitted with a flame ionisation detector and a porapak column. The algal pellet was digested in 5 ml of 0·5 Ν NaOH to estimate protein following Lowry et al. (1951). Nitrogenase activity is expressed in terms of nmol of ethylene formed per mg protein per hour. Results and discussion Growth The growth of Anabaena ARM310 was measured in terms of chlorophyll a content (figure 1). In the presence of phosphates, soluble or insoluble, growth was greater than in the absence of phosphates. The growth observed in the present study indicates that the organism could utilise extracellular insoluble phosphates under both CN+ and CN– conditions. A number of other cyanobacteria, such as Anabaena, Nostoc, Tolypothrix, Aulosira and Anacystis, have been shown to solubilize extracellular insoluble phosphates (Bose et al., 1971).
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Figure 1. Growth of Anabaena ARM310. , Chlorophyll a content of the initial inoculum; (
), chlorophyll a content of cells at the end of the growth period.
Phosphate solubilization The pH of the culture filtrate containing different phosphate sources with and without combined nitrogen showed marginal increase over the respective uninoculated control (table 1). Earlier reports in bacteria indicate acid hydrolysis by Table 1. pH of the culture filtrates of Anabaena ARM310.
Data represent average of two estimations. UC, Uninoculated control. Exp., Experimental. CN+, Medium with combined nitrogen; CN–, medium without combined nitrogen; ISP+, medium with tricalcium phosphate; SP+, medium with dipotassium hydrogen phosphate; SP–, medium without dipotassium hydrogen phosphate.
organic acids as a possible mechanism of phosphate solubilization (Subba Rao, 1982). Sperber (1958) reported that acids secreted by the organisms lowered the pH
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of the medium and thereby dissolved the bound phosphates. However, in the present study this does not seem to happen since the pH of the culture filtrate never dropped to the acidic range. Under conditions of phosphate starvation cyanobacteria are known to show increased levels of intracellular and cell surface alkaline phosphatase activity to solubilize polyphosphates (Healey, 1982). There was a shift in the pH towards alkalinity in all the inoculated treatments while in the uninoculated treatments the pH remained unchanged. This observation prompted us to investigate the involvement of alkaline phosphatases in solubilization of extracellular phosphates. Under conditions of soluble phosphate depletion and insoluble phosphate addition (figure 2A,C), alkaline phosphatase activity was observed from the 10th
Figure 2. Alkaline phosphatase activity in the culture filtrate of Anabaena ARM310.
day, which increased on the 15th and 20th day. However, in the presence of soluble phosphates the activity was detected only from the 15th day (figure 2B). This lends support to the argument that alkaline phosphatases are induced and secreted on phosphate starvation/depletion and that these are involved in the solubilization of tri-calcium phosphate.
Figure 3. Acetylene reduction assay of Anabaena ARM310. Nitrogenase activity of the cells on 10th(
) and 15th day.
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Acetylene reduction activity There was no significant difference in the acetylene reduction activity of the cells incubated in treatments Ε and F (figure 3). The cyanobacterium Anabaena ARM310 seems to be an ideal choice for the preparation of microbial inoculant because of its ability to grow well and fix atmospheric nitrogen in the absence of combined nitrogen and soluble phosphates. Acknowledgements One of the authors (R.N.) was supported by the University Grants Commission's FIP. Funds provided from the Indian Council of Agricultural Research, New Delhi, under the Indo-US project is acknowledged with thanks. References Bardiya, M. C. and Gaur, A. C. (1972) Indian J. Microbiol, 12, 269. Bose, P., Nagpal, U. S., Venkataraman, G. S. and Goyal, S. K. (1971) Gurr. Sci., 40, 165. Freeland, P. W. (1985) Problems in practical advanced level biology (England: Hodder and Stoughton) Gaur, A. C. (1988) Mycorrhiza round table (Canada: IDRC and New Delhi: Jawaharlal Nehru University) Healey, F. P. (1982) in Biology of the cyanobacteria (eds N. G. Carr and B. A. Whitton) (Oxford: Blackwell) p. 105. Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem., 93, 265. Mackiney, G. (1941) J. Biol. Chem., 140, 315. Rippka, R., Deruelles, J., Waterbury, J. Β., Herdman, Μ. and Stanier, R. Y. (1979) J. Gen. Microbiol., 111, 1. Sperber, J. I. (1958) Nature (London), 181, 934. Stewart, W. D. P., Fitzgerald, G. P. and Burris, R. H. (1968) Arch. Microbiol, 62, 336. Subba Rao, N. S. (1982) Biofertilizers in agriculture 2nd edition (New Delhi: Oxford and IBH Publ. Co.) Venkataraman, G. S. (1972) Algal biofertilizers and rice cultivation (New Delhi: To-day and Tomorrow's Printers and Publishers). Wani, P. V., More, B. B. and Patil, L. K. (1980) Macco Agric. Digest, 4, 11.
