Phosphate solubilizing potential of Aspergillus niger MPF-8 isolated from Muthupettai mangrove

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1 Journal of Scientific & Industrial Research Vol. 74, September 2015, pp Phosphate solubilizing potential of Aspergillus niger MPF-8 isolated from Muthupettai mangrove S Bhattacharya 1 *, A Das 1, S Bhardwaj 2 and S S Rajan 3 *1 Department of Microbiology, Center for Post Graduate Studies, Jain University, Bangalore , Karnataka, India 2 Department of Biotechnology, The Oxford College of Science, Bangalore , Karnataka, India 3 Department of Microbiology, Centre for Advanced Studies in Biosciences, Jain University, Bangalore , Karnataka, India Received 16 February 2014; revised 19 December 2014; accepted 11 July 2015 The present study deals with the isolation of fungi from the mangrove sediment and determining their tri-calcium phosphate solubilization efficiency. Among the 47 fungal isolates, MPF-8 showed maximum phosphate solubilization and based upon molecular identification using 18S rdna sequencing was identified as Aspergillus niger. Among the various carbon and nitrogen sources added to Pikovskaya broth, maximum phosphate solubilization, 401 µg/ml and 427 µg/ml were recorded with glucose and ammonium sulphate supplementation, respectively. Optimum ph and temperature for maximum phosphate solubilization was 7.0 and 30 C, liberating 443 µg/ml and 468 µg/ml of soluble phosphate, respectively. Following incubation, a reduction in the ph of the medium was recorded at all stages of the optimization process, indicating the involvement of organic acids in the solubilization process. These results emphasized that the mangrove isolate of A. niger MPF-8 may be utilized as a biofertilizer for solubilization of soil-bound phosphates, thereby increasing the soil fertility. Keywords: Aspergillus niger, mangrove, tri-calcium phosphate, phosphate solubilization Introduction Mangroves are a diverse group of plants dominating the majority of world's tropical and subtropical coastline and are an ecological entity with little phylogenetic association. Over millions of years, these plants have evolved both morphologically and physiologically to adapt to swampy and saline environments. Similar adaptive characteristics in the form and function may occur with the associated microflora (bacteria, fungi, algae, etc) in such environments 1.Plant mineral nutrition depends mainly on the phosphorus content of soil, which can be assimilated only as soluble phosphate. However, in mangrove soils, phosphate remains immobilized and unavailable for plant use. Therefore, in such nutrientlimited environments, organisms that solubilize phosphate can have important implications for plant growth. Rhizospheric phosphate solubilizing microorganisms (PSMs), mainly few soil bacteria, fungi, yeasts and actinomycetes are involved in solubilization of soil bound insoluble phosphates. Symbiotic associations between roots and fungi are widespread in nearly all soil types and are important for the uptake of Author for correspondence bhattsourav3011@gmail.com immobile nutrients, especially for the solubilization of phosphorus 2. Several mechanisms like lowering of ph by secreting weak organic acids, production of acid and alkaline phosphatases, ion chelation and exchange reactions are involved in the process 3. Among PSMs, several species of filamentous fungi and yeasts have been reported to solubilize different forms of inorganic phosphates 4. In the present study, fungal strains were isolated from Muthupettai mangrove ecosystem and checked for their phosphate solubilizing potential under different cultural conditions. Materials and Methods Sample collection Sediment sample was collected by inserting a stainless steel corer sampler (inner diameter 32 mm) into the sediment near the root system of Avicennia marina from Muthupettai mangrove forest (10 24 N, E). Following sampling, the central portion of the 20 cm sediment sample was collected in zip-lock cover and transported to the laboratory. Isolation and screening of phosphate solubilizing fungi Soil sample (1 g) was serially diluted and spread plated on potato dextrose agar (PDA) for the isolation

2 500 J SCI IND RES VOL 74 SEPTEMBER 2015 of rhizospheric fungi. Following the appearance of fungal colonies on agar medium, these isolates were screened for their phosphate solubilizing ability by point inoculation on Pikovskaya (PVK) agar supplemented with 0.5% (w/v) tri-calcium phosphate (Merck, Germany). After 5 days of incubation at 28±2 C, formation of a halo around the fungal growth on PVK agar indicated phosphate solubilization. The degree of phosphate solubilization was determined by measuring the clear zone (in mm) around the colonies. Molecular identification of the selected isolate The culture demonstrating the highest zone of phosphate solubilization was identified using 18S rdna sequencing (Chromous Biotech Pvt. Ltd., Bangalore, India). Forward and reverse 18S rdna primers used were (5 -GTAGTCATATGCTTGTCTC-3 ) and (5 -GAAACCTTGTTACGACTT-3 ), respectively. The fungal specie was identified by comparing the obtained sequence with known 18S ribosomal sequences in the NCBI database using BLASTN 5. The nucleotide sequence was submitted to GenBank database (NCBI, USA) and was provided an accession number. Preparation of inoculum Spore suspension was prepared by mixing a loopful of fungal spores in 10 ml of sterile distilled water. A uniform spore suspension was obtained by mixing vigorously, which was measured for absorbance under white light. 1 ml of spore suspension (10 7 spores/ml) was used as inoculum. Optimization of cultural conditions PVK broth (50 ml, ph 6.5) was supplemented with 0.5% (w/v) tri-calcium phosphate. In order to determine the effect of different nutrient supplements on phosphate solubilization, the above broth was amended with various sugars (1% w/v: glucose, fructose, lactose, maltose, sucrose, soluble starch and xylose), organic and inorganic nitrogen sources (1% w/v: peptone, tryptone, yeast extract, beef extract, ammonium sulphate, urea and sodium nitrate). The flasks were incubated at 28±2 C for 10 days at 120 rpm. Various physical parameters such as initial ph of the medium (4, 5, 6, 7, 8, 9 and 10) and incubation temperature (20, 25, 30, 35, 40, 45 and 50 C) were also optimized. In the above cases, the uninoculated broth served as control. All the experiments were conducted in triplicates. Analytical technique Following incubation, the contents of the flasks (including the control) were centrifuged at 5000 rpm for 20 min at 4 C. The cell free supernatant was filtered through 0.22 μ membrane filter and the clear filtrate was analyzed for final ph and quantitative amount of soluble phosphate 6. Dissolved phosphate concentration in the culture filtrate was expressed in terms of μg/ml phosphorus released in culture medium. Statistical analysis All the optimization studies were conducted in triplicates and the data were analyzed using single factor analysis of variance (ANOVA). All the data are graphically presented as mean ± standard deviation of triplicates (n=3). ANOVA was performed using Microsoft Excel P values < 0.05 were considered significant with a confidence limit of 95%. Results and Discussion Dissolved inorganic phosphate exists in mangrove soil mainly as ionic forms of orthophosphoric acid. Since the phosphate ions are negatively charged, they are quickly absorbed after the weathering of clays or detritus particles, thus forming insoluble forms of aluminium, calcium or iron phosphates, all unavailable to mangroves 7. Microorganisms in mangroves play an important role in recycling of nutrients and establishment of mangrove forests through mineralization and also by production of phytohormones 8. Screening of phosphate solubilizing fungal isolates Of the total 47 fungal isolates, 6 demonstrated significant solubilization of insoluble tri-calcium phosphate when grown on PVK agar medium and were designated as MPF-2, 8, 13, 18, 31 and 39. Various degrees of phosphate solubilization were observed and the highest zone of clearance (11 mm) was demonstrated by isolate MPF-8, which was selected for further study. Molecular identification of the selected isolate 18S rdna sequencing is a powerful tool for rapid identification and phylogenetic analysis of fungal species. The obtained 550 bp 18S rdna nucleotide sequence was compared with available 18S ribosomal sequences in the NCBI database using BLASTN. The MPF-8 isolate has been enrolled into a cluster containing Aspergillus sp. and was found to be closely related to Aspergillus niger strain HPA8 with 99%

3 BHATTACHARYA et al.: PHOSPHATE SOLUBILIZING POTENTIAL OF ASPERGILLUS NIGER 501 sequence similarity. Hence it was designated as Aspergillus niger MPF-8. The submitted nucleotide sequence was provided a GenBank accession number KM Optimization of carbon source Microorganisms ferment the carbon source available in the medium to produce several organic acids. In the process of phosphate solubilization, these acids play a significant role to convert the insoluble phosphates to the available form, where the nature of acid(s) produced is more important than the quantity of the acid(s) 9.The present study demonstrated that the extent of phosphate solubilization was related to the nature of the particular carbon supplement in the medium. Phosphate solubilization by A. niger MPF-8 was maximum (401 µg/ml) in PVK broth supplemented with glucose (Fig. 1). This particular medium also demonstrated maximum accumulation of acid following incubation. Our results are in agreement with the reports of previous researchers who found that in case of Aspergillus sp., glucose and maltose decreased the ph of the medium to maximum extent and caused highest solubilization of phosphorus. This was followed by sucrose, xylose and galactose 10. Goenadi et al. 11 reported better solubilization of phosphate by A. niger using glucose as the carbon source.among the carbon sources, A. niger BHUAS01 solubilized maximum tri-calcium phosphate (512 μg/ml) in the presence of glucose followed by glycerol, maltose and sucrose after 21 days of incubation in PVK broth 12. A study investigating the effect of carbon and nitrogen sources on phosphate solubilization by a wild strain of A. tubingensis, reported significant phosphate solubilization in the presence of glucose and sucrose as compared to fructose, lactose, galactose and xylose 13.On the contrary, A. niger F7 demonstrated diverse levels of phosphate solubilization in the presence of various carbon sources in the order: maltose (420 μg/ml) > sucrose (307.5 μg/ml) > glucose (300 μg/ml) accompanied by a drop in ph from 7.0 to 3.35, 3.52 and 3.47, respectively 9. Optimization of nitrogen source A number of fungi had been reported to solubilize phosphate only in the presence of ammonium salts as nitrogen source 10. Among the nitrogen sources used, ammonium sulphate was found to be the best, resulting in solubilization of 427 μg/ml of phosphorus and simultaneous reduction of the medium ph to 4.14 (Fig. 2). Acid production in response to the assimilation of cations such as ammonium is a well known fungal phenomenon. The uptake of ammonium ions by fungi in a liquid medium commonly leads to a rapid drop in ph of the medium. Similar to the present result, more than the organic sources of nitrogen, ammoniacal nitrogen sources were reported to increase the solubilization of fluorapatite by A. niger 14. Likewise, it was observed that A. niger F7 was able to utilize ammonium sulphate and ammonium chloride most efficiently to decrease the ph of the medium for phosphate solubilization 9.A. aculeatus solubilized phosphate from tri-calcium phosphate in PVK medium and the phosphate solubilization was highest after 48 h of fungal growth in the presence of glucose and ammonium sulphate as the best carbon and nitrogen sources, respectively 15. Effect of initial ph of the medium on phosphate solubilization Microbial cells are significantly affected by the ph of their immediate environment because they apparently have no mechanism for adjusting their Fig. 1 Effect of carbon supplements on phosphate solubilization by A. niger MPF-8. Data represent mean ± S.D. (n=3); P < 0.05 Fig. 2 Effect of nitrogen supplements on phosphate solubilization by A. niger MPF-8. Data represent mean ± S.D. (n=3); P < 0.05

4 502 J SCI IND RES VOL 74 SEPTEMBER 2015 internal ph 16. Therefore in the present study, the effect of initial ph of the medium on phosphate solubilization was investigated.when tested for the ability to solubilize inorganic phosphate at different ph, the initial ph of the culture medium influenced phosphate solubilization by A. niger MPF-8. Solubilization was highest (443 μg/ml) in the medium with neutral ph in comparison to those having extremes of acidic and alkaline ph (Fig. 3). Similarly, among the phyllosphere fungi isolated from Bhitarkanika mangrove plants, Aspergillus PF8 when grown on tri-calcium phosphate supplemented medium at ph 4.5, 7.0 and 9.0 showed maximum clearing zones of phosphate solubilization (47.5 mm and 63 mm) at ph 7.2, 30 C and 37 C 17.However, at initial ph of 5.5 A. niger released maximum content of soluble phosphate (89.3 mg/l). At ph values higher or lower than the optimal initial ph, the content of soluble phosphorus decreased 18. Effect of incubation temperature on phosphate solubilization Different levels of phosphate solubilization were recorded when A. niger MPF-8 was incubated at various temperatures. Maximum phosphate solubilization (468 µg/ml) was observed at 30 C followed by 35 C (419 µg/ml), 25 C (402 µg/ml), 20 C (334 µg/ml), 45 C (309 µg/ml) and 50 C (211 µg/ml), respectively. An increase in incubation temperature also influenced the reduction in the ph of the culture medium. The present finding is in accordance with that of a previous study wherein 30ºC resulted in maximum phosphate solubilization (78.63%) by A. niger PSF4 19. Similarly, other workers found 30ºC to be the best temperature for phosphate solubilization by A. niger 18.Although many researchers reported phosphate solubilization at different temperatures, most of them suggested Fig. 3 Effect of initial ph of medium on phosphate solubilization by A. niger MPF-8. Data represent mean ± S.D. (n=3); P < ºC to be the optimum temperature for phosphate solubilization 20,21. Conclusions The mangrove isolate A. niger MPF-8 proved to be a potential phosphate solubilizing fungus. Furthermore, the ability of A. niger MPF-8 to convert insoluble tri-calcium phosphate to soluble form was greatly enhanced by the incorporation of glucose and ammonium sulphate in the medium maintained at initial neutral ph and incubation temperature of 30 C. The results obtained in the present study suggest that A. niger MPF-8 may be subjected to field applications where it can act as a biofertilizer, thus helping in increased nutrient uptake by plants. Acknowledgements We wish to extend our sincere gratitude to the management of Jain University for its encouraging support. References 1 Gupta N, Das S & Basak U C, Useful extracellular activity of bacteria isolated from Bhitarkanika mangrove ecosystem of Orissa coast, Mal J Microbiol, 3 (2007) Smith S E, Smith F A & Jakobsen I, Mycorrhizal fungi can dominate phosphate supply to plants irrespective of growth responses, Plant Physiol, 133 (2003) Achal V, Savant V V & Reddy M S, Phosphate solubilization by a wild type strain and UV indued mutants of Aspergillus tubingensis, Soil Biol Bichem, 39 (2007) Das A, Dutta B K & Barooah A K, In vitro solubilization of inorganic phosphate by phosphate solubilizing fungi isolated from tea agrosystem soil of Barak Valley, Southern Assam, Int J Microbiol Res, 4 (2013) Altschul S F, Gish W, Miller W, Myers E W & Lipman D J, Basic local alignment search tool, J Mol Biol, 215 (1990) Dick W A & Tabatabai M A, Determination of orthophosphate in aqueous solutions containing labile organic and inorganic phosphorus compounds, J Environ Qual, 6 (1977) Vazquez P, Holguin G, Puente M E, Lopez-Cortes A & Bashan Y, Phosphate-solubilizing microorganisms associated with the rhizosphere of mangroves in a semiarid coastal lagoon, Biol Fertil Soils, 30 (2000) Bhardwaj S, Bhattacharya S & Das A, Phosphate solubilizing activity of a mangrove isolate of Streptomyces badius from Muthupettai mangrove, Tamil Nadu, India, J Chem Biol Phys Sci, 2 (2012) Srividya S, Soumya S & Pooja K, Influence of environmental factors and salinity on phosphate solubilization by a newly isolated Aspergillus niger F7 from agricultural soil, Afr J Biotechnol, 8 (2009) Pradhan N & Shukla L B, Solubilization of inorganic phosphates by fungi isolated from agriculture soil, Afr J Biotechnol, 5 (2005) Goenadi D H, Siswanto & Sugiarto Y, Bioactivation of poorly soluble phosphate rocks with a phosphorussolubilizing fungus, Soil Sci Soc Am J, 64 (2000)

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