Directory UMM :Data Elmu:jurnal:S:Soil Biology And Chemistry:Vol32.Issue4.2000:

Aspergillus aculeatus

as a rock phosphate solubilizer

Varsha Narsian, H.H. Patel*

Department of Life Sciences, Bhavnagar University, Bhavnagar, Gujarat 364 002, India

Accepted 28 September 1999

Abstract

Aspergillus aculeatus, a rhizosphere isolate of gram (Cicer ariatenum) invariably solubilized both foreign (China and Senegal) and Indian (Hirapur, Udaipur, Sonrai) rock phosphates (RP). Arabinose proved superior to glucose among 11 carbon compounds tested. All the test carbon sources, except lactose, supported good growth regardless of their phosphate solubilization. Ammonium sulphate was the best nitrogen source followed by urea and asparagine. Nitrates, while supporting good growth, did not support good phosphate solubilization. Maximum phosphate solubilization (PS), growth and acidity of the medium never occurred simultaneously with all C-to-N ratios (8 to 120). The chelator nitrilotriacetic acid increased RP solubilization at 2 mg mlÿ1

while diethylenetriaminepentaacetic acid enhanced PS only at 6 mg mlÿ1 , while ethylenediaminetetraacetic acid, aluminon and oxine inhibited PS at all concentrations tested. Although increasing concentration of RP in the culture medium favoured mycelium growth and a decrease in pH, soluble phosphate concentrations were reduced, probably owing to consumption by the rapidly growing fungus. Acidic pH accompanied PS in all the experiments. However, no speci®c correlation could be established between maximum PS, growth or pH.72000 Elsevier Science Ltd. All rights reserved.

Keywords:Rock phosphate; Phosphate solubilization; Carbon sources; Nitrogen sources; Chelators;Aspergillus aculeatus

1. Introduction

Phosphorus is added to soil in the form of phospha-tic fertilizers, part of which is utilized by plants and the remainder converted into insoluble ®xed forms. Phosphate solubilizing microorganisms (PSM) render these insoluble phosphates into soluble form through the process of acidi®cation, chelation and exchange reactions. Microbial solubilization of rock phosphate (RP), especially low grade and its use in agriculture is receiving greater attention. This process not only com-pensates for higher cost of manufacturing fertilizers in industry but also mobilizes the fertilizers added to soil. A few workers reported RP solubilization in liquid medium by various fungi (Meyer and Konig, 1960; Ahmed and Jha, 1968; Gaur et al., 1973; Bardiya and Gaur, 1974; Khan and Bhatnagar, 1977; Arora and Gaur, 1979; Singh et al., 1984; Sattar and Gaur, 1985).

In the present work,Aspergillus aculeatus was exam-ined for its RP solubilizing ability and some factors a€ecting RP solubilization.

2. Materials and methods

2.1. Organism

A. aculeatus was isolated from the rhizosphere of gram (Cicer ariatenum) by the method described by Gaur (1987). The culture was maintained on agar slants described by Pikovskaya (1948).

2.2. Culture medium

In Pikovskaya's broth, tricalcium phosphate (TCP) was replaced with di€erent RPs, 2 foreign, Senegal (SRP, 36.7 mg% P2O5) and China (CRP, 33 mg% P2O5) and 3 Indian RPs, Hirapur (HRP, 26.4 mg% P2O5), Sonrai (RRP, 27.5 mg% P2O5) and Udaipur

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(URP, 27.6 mg% P2O5) in the amount equivalent to 50mg P2O5100 mlÿ1.

E€ect of di€erent carbon sources on growth, phos-phate solubilizing (PS) activity and change in the pH of the medium (TCP was replaced with RRP=50 mg 100 mlÿ1 _P

2O5) was observed by replacement of glu-cose with 10 individual carbon compounds sterilized separately and added to the medium before inocu-lation. The nitrogen sources were evaluated similiarly by replacing ammonium sulphate in the medium with six di€erent nitrogen sources. The e€ect of varying C-to-N ratio was studied by adjusting the amount of C and N compounds in the medium such that the ratio attained was 8,10,...,120.

A set of experiment was conducted to study the e€ect of chelation on P-solubilization. Five di€erent chelators, viz. Ethylenediaminetetraacetic acid (EDTA), Diethylenetriaminepentaacetic acid (DTPA), Nitrilotriacetic acid (NTA), aluminon and oxine were added at concentrations from 1±6 mg mlÿ1_in

Pikovs-kaya's medium containing RRP=50 mg P2O5 100 mlÿ1

.

In another set, TCP in the medium was replaced by RRP equivalent to 25, 50, 75, 100, 150, 200 and 250 mg P2O5100 mlÿ1for studying the e€ect of increasing concentration of RP on P-solubilization by A. aculea-tus.

All the experiments were carried out in triplicate.

2.3. Inoculation and growth condition

All the aforesaid set of media were inoculated each with 8 mm mycelial discs (75106 spores mlÿ1) from 4 d old culture of A. aculeatus grown on Czapek's Dox agar. All sets of ¯asks were incubated at 308C for 15 d and shaken at 12 h intervals. Uninoculated ¯asks from each set were treated similarly for control.

2.4. Growth determination

At the end of incubation, the contents of the ¯asks

were ®ltered through Whatman No. 42 ®lter paper, washed repeatedly with distilled water and the mycelial mat dried at 988C overnight. Dry weight of the my-celium represented the growth.

2.5. Phosphorus estimation and pH

Water soluble P in the culture ®ltrate was estimated after periodic intervals up to 15 d by the chlorostan-nous reduced molybdophosphoric acid blue method described by Jackson (1967). The pH of the spent med-ium was measured by a pH meter.

Statistical interpretation of the various measure-ments made were carried out using analysis of variance (ANOVA) and other standard methods outlined by Snedecor and Cochrane (1967).

3. Results and discussion

A. aculeatus was capable of solubilizing all natural forms of phosphorus tested i.e. both foreign and Indian RPs (Table 1). The same organism maximally solubilized TCP (107.40 mg% P2O5) on d 2 (Narsian et al., 1995). However, RPs were solubilized to a lesser extent than TCP. The results (Table 1) of the periodic solubilization of di€erent RP showed that the maxi-mum solubilization of CRP and URP was achieved earlier i.e. by d 8 while SRP, RRP and HRP was on d 14. The maximum amount of P2O5released byA. acu-leatus from CRP, SRP, RRP, HRP and URP were 91.6, 28.4, 46.2, 40.7 and 38.1% respectively. Aspergil-lus awamori also solubilized URP (46.8 mg%) which proved better than A. aculeatus while Penicillium digi-tatum (28.12 mg%) showed lesser solubilization of URP (Gaur, 1990).

While comparing the relative eciency of A. aculea-tususing di€erent RP, it was observed that RP solubil-ization depended on the nature of RP and the organism. The relative eciency ofA. aculeatusfor RP solubilization may have been due to the nature and

Table 1

Release of soluble P from di€erent forms of native rock phosphates byA. aculeatus

Phosphorus sources Time (d)

3 5 8 14

P2O5a(mg%) pH P2O5(mg%) pH P2O5(mg%) pH P2O5(mg%) pH

China rock phosphate 15.43 5.0 25.28 5.7 45.80 5.6 7.69 7.2

Senegal rock phosphate 8.88 4.0 5.99 6.5 7.87 6.2 14.19 6.2

Sonrai rock phosphate 5.45 3.7 3.93 6.5 16.57 6.6 23.08 5.6

Hirapur rock phosphate 7.09 3.6 5.99 6.4 9.89 6.4 20.33 6.2

Udaipur rock phosphate 1.23 4.0 5.99 5.0 19.05 6.1 17.77 6.0

Table 2

E€ect of di€erent carbon and nitrogen sources on Sonrai RP solubilization byA. aculeatus

Mycelial dry wt. (MDW) (mg mlÿ1

) Final pH of the medium Maximum P solubilized as P2O5(mg%) Carbon source

Arabinose (Ara) 5.01 3.5 28.39

Glucose (Glu) 5.12 5.3 23.08

Fructose (Fru) 7.01 4.3 18.22

Mannitol (Man) 5.01 3.5 10.85

Xylose (Xyl) 6.36 3.5 9.52

Maltose (Mal) 7.22 3.3 8.28

Sorbitol (Sor) 7.48 5.0 6.54

Sucrose (Suc) 6.88 4.5 4.44

Glycerol (Gly) 6.44 4.8 3.93

Galactose (Gal) 6.12 6.0 2.56

Lactose (Lac) 4.04 5.0 1.64

Nitrogen source

(NH4)2SO4(AS) 5.12 5.6 23.08

Asparagine (ASP) 5.80 3.7 18.22

Urea (Urea) 7.84 6.8 13.01

Ca(NO3)2(CN) 8.26 4.2 10.85

NaNO3(SN) 7.68 4.5 7.69

NH4NO3(AN) 7.40 5.9 6.54

KNO3(PN) 8.04 5.7 5.99

quantity of organic acids secreted in the medium. However, more work is required regarding the nature of RPs and their interaction with ecient PSM.

A decrease in the pH of the medium with all P-sources tested was invariably observed. The lowest pH was recorded in the presence of HRP (pH 3.6) while the highest pH in the presence of CRP (pH 7.2) which may be due to complexity of the RP. However, no sig-ni®cant relationship could be established between the quantity of phosphate solubilized and drop in pH. This ®nding is in agreement with reports of Ahmed and Jha (1968), Das (1963), Sethi and SubbaRao (1968) and Sperber (1958).

To determine whether phosphate solubilization was a€ected by various carbon sources, A. aculeatus was grown in medium containing di€erent C-sources (Table 2). A. aculeatus could use di€erent carbon sources but preference, irrespective of days in relation to highest PS activity was in the following order: ara-binose > glucose > fructose > mannitol > xylose > maltose > sorbitol > sucrose > glycerol > galactose > lactose. However, all carbon sources except lactose

improved growth relative to glucose. This, however does not indicate a clear cut relationship between growth and PS activity. A decrease in the pH of the medium was always observed.

Fig. 1 illustrates the temporal correlation between PS activity, acidity and growth in presence of RRP by

A. aculeatus. Highest PS activity, acidity and growth occurred at the same time for sucrose, maltose, lactose and glycerol while a positive correlation between high-est PS activity and greater acidity was noted for xylose and mannitol. A direct correlation between PS activity and growth for glucose, fructose, galactose, arabinose, xylose and mannitol was not observed.

The test fungus could grow and solubilize large amounts of RRP using di€erent forms of nitrogen (Table 2). Ammonium sulphate was the best source of N followed by organic forms, urea and asparagine. Although larger amounts of phosphate were solubil-ized by ammonium sulphate and asparagine, greater growth was observed with the remaining test nitrogen sources. pH was also reduced invariably. As noted in the case of carbon sources, the relationship between

PS activity with growth or pH could not be deter-mined.

Fig. 2 shows the temporal correlation between high-est PS activity, acidity of the medium and growth of

A. aculeatus with RRP as P source. RRP solubil-ization, acidity and growth did not occur simul-taneously. There was no correlation between highest PS activity and acidity with all test nitrogen sources. Also no correlation was noted between highest PS ac-tivity and growth when ammonium sulphate, am-monium nitrate and asparagine served as nitrogen source.

The C-to-N ratio, besides absolute carbon and nitro-gen contents, greatly modi®ed the PS activity ruling on both acidity and growth. Keeping this in mind, we have attempted to derive from the data (Fig. 3), the temporal correlation between PS activity, growth and acidity of the medium with di€erent C-to-N ratios in the presence of RRP. As in the case of N sources, highest PS activity, growth and acidity never occurred simultaneously with all C-to-N ratios. However, simi-larly no correlation was observed between all C-to-N ratios for the highest PS activity and highest growth.

A positive correlation for the highest PS activity and greater acidity was observed with C-to-N ratios 8, 20 and 60.

Phosphate solubilization has been attributed to the process of acidi®cation and chelation by many workers (Agnihotri, 1970; Molla and Chowdhary, 1984; Kucey, 1987; Asea et al., 1988). Keeping this in mind, the e€ect of A. aculeatus on the action of chelators was studied for its e€ect on PS activity.

On studying the e€ect of increasing concentration of various chelators (EDTA, DTPA, NTA, aluminon and oxine) on RRP solubilization, test chelators had di€er-ential behaviours in relation to phosphate solubil-ization (Fig. 4). NTA increased RP solubilsolubil-ization at 2 mg mlÿ1

concentration while DTPA had an enhancing e€ect only at 6 mg mlÿ1

concentration. The other three viz. EDTA, aluminon and oxine showed inhi-bition of PS activity at all concentrations (1±6 mg mlÿ1

). The action of chelators appears to be due to functional groups, such as carboxylic or phenolic hy-droxyls.

The microbial solubilization of RRP with increasing concentration of P is shown in Fig. 5. Except for

Fig. 4. Sonrai rock phosphate solubilization on d 14 in presence of RP and di€erent concentration of chelators byA. aculeatus.

centrations 50 and 100 mg, all concentrations showed their highest PS activity on d 5. Increasing concen-tration of P accompanied increased growth of fungi up to d 14. Results revealed that the highest PS activity, in presence of RP, was up to 50 mg P2O5. Higher con-centrations than this did not show any further activity; in fact the activity of the fungus was considerably reduced with further concentration (i.e. from 100 to 250 mg P2O5). Although higher concentrations of P were not e€ective for PS activity, growth, however, increased successively.

Correlation studies (Table 3) with the P solubil-ization and change in pH showed a perfect positive re-lationship with URP, moderate rere-lationship for RRP and HRP and an inverse relationship with CRP. How-ever, no correlation could be established between pH and PS activity in the presence of HRP. An inverse correlation was noted with carbon sources between PS activity and pH and PS activity and dry weight. A moderate positive relation was noted between PS ac-tivity and dry weight while an inverse relation between PS activity and pH with nitrogen sources. In the case of C-to-N ratios, a moderate association was noted between PS activity and pH while an inverse relation between PS activity and dry weight. A similar inverse relation between PS activity and dry weight was noted with di€erent concentrations of rock phosphates.

It was also observed that the medium receiving the lowest quantity of RP remained more acidic than media with higher concentrations of P. This agrees with the observation of Gaur (1990).

References

Agnihotri, V.P., 1970. Solubilization of insoluble phosphates by some soil fungi isolated from nursery seedbeds. Canadian Journal of Microbiology 16, 877±880.

Ahmed, N., Jha, K.K., 1968. Solubilization of rock phosphate by microorganisms isolated from Bihar soils. Journal of Canadian Applied Microbiology 14, 89±95.

Arora, D., Gaur, A.C., 1979. Microbial solubilization of di€erent inorganic phosphates. Indian Journal of Experimental Biology 17, 1258±1261.

Asea, P.E.A., Kucey, R.M.N., Stewart, J.W.B., 1988. Inorganic phosphate solubilization by two Penicillium species in solution culture and soil. Soil Biology & Biochemistry 20, 459±464. Bardiya, M.C., Gaur, A.C., 1974. Isolation and screening of

micro-organisms dissolving low grade rock phosphate. Folia Microbiologiya 19, 386±389.

Das, A.C., 1963. Utilization of insoluble phosphate by soil fungi. Journal of Indian Society of Soil Science 11, 195±207.

Gaur, A.C., Madan, M., Ostwal, K.P., 1973. Solubilization of phos-phate compounds by native micro¯ora of rock phosphos-phates. Indian Journal of Experimental Biology 11, 427±429.

Gaur, A.C., 1987. Phosphorus solubilizing biofertilizers. In: Gaur, A.C. (Ed.), Organic Manures and Biofertilizers. Indian Agricultural Research Institute, New Delhi, pp. 46±65.

Gaur, A.C., 1990. Phosphate Solubilizing Microorganisms as Biofertilizer. Oxford Publishing Co, New Delhi, pp. 26±29. Jackson, M.L., 1967. Soil Chemical Analysis. Prentice Hall of India,

New Delhi, pp. 144±145.

Khan, J.A., Bhatnagar, R.M., 1977. Studies on solubilization of insoluble phosphates by microorganisms. Part I. Solubilization of Indian phosphate rocks byA. nigerandPenicilliumsp. Fertilizer Technology 14, 329±333.

Kucey, R.M.N., 1987. Increased phosphorus uptake by wheat and ®eld beans inoculated with a phosphorus solubilizingPenicillium bilaji strain and with vesicular-arbuscular mycorrhizal fungi. Applied and Environmental Microbiology 53, 2699±2703. Meyer, L., Konig, E., 1960. Experimental results on the biological

decomposition of dicultly soluble phosphate by soil fungi. Landwirtschaftliche Forschung 13, 7±24.

Molla, H.A.Z., Chowdhary, A.A., 1984. Microbial mineralization of organic phosphate in soil. Plant and Soil 78, 393±399.

Narsian, V., Thakkar, J., Patel, H.H., 1995. Mineral phosphate solu-bilization by A. aculeatus. Indian Journal of Experimental Biology 33, 91±93.

Pikovskaya, R.I., 1948. Mobilization of phosphorus in soil in con-nection with vital activity of some microbial species. Mikrobiologiya 17, 362±370.

Sattar, M.A., Gaur, A.C., 1985. Characterization of phosphate dis-solving microorganisms isolated from some Bangladesh soil samples. Bangladesh Journal of Microbiology 2, 22±28.

Sethi, R.P., SubbaRao, N.S., 1968. Solubilization of tricalcium phos-phate and calcium phosphos-phate by soil fungi. Journal of General and Applied Microbiology 14, 329±331.

Singh, H.P., Pareek, R.P., Singh, T.A., 1984. Solubilization of rock phosphate by phosphate solubilizers in broth. Current Science 53, 1212±1213.

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Table 3

Correlation of soluble phosphate with pH and mycelial dry weight P2O5(mg 100 mlÿ

China rock phosphate ÿ0.44 ±

Senegal rock phosphate 0.00 ±

Sonrai rock phosphate 0.26 ±

Hirapur rock phosphate 0.32 ±

Udaipur rock phosphate 0.99 ±

Carbon sources ÿ0.28 ÿ0.17

Nitrogen sources ÿ0.14 0.35

C-to-N ratios 0.38 ÿ0.29