ORIGINAL ARTICLE

Solubilization of inorganic phosphates by Aspergillus awamoriS19 isolated from rhizosphere soil of a semi-arid region

Rachana Jain & Jyoti Saxena & Vinay Sharma

Received: 18 March 2011 /Accepted: 27 June 2011 /Published online: 12 July 2011# Springer-Verlag and the University of Milan 2011

Abstract A phosphate solubilizing fungus, Aspergillusawamori S19, was isolated from the rhizosphere soil ofPennisetum glaucum grown in semi-arid climatic condi-tions. The organism was identified on the basis ofmorphological characterization and by sequencing ofITS1-5.8S-ITS2 region. The organism was able to solubi-lize various inorganic forms of phosphate at a wide range oftemperatures. Amongst various insoluble phosphate sourcestested, di-calcium phosphate (DCP) was solubilized themost, followed by tri-calcium phosphate (TCP). Thesolubilization was evident at all the temperatures but theperformance was especially good in the range of 25–35°C.Phosphate-solubilizing ability was also measured in thepresence of various carbon and nitrogen sources. Maltosewas found to be the best carbon source, followed byglucose, whereas, amongst nitrogen sources, ammoniumchloride supported maximum solubilization, followed byammonium sulphate, sodium nitrate and urea. Ammoniumrather than nitrate was found to be more promising as thesole source of nitrogen for phosphate solubilization. Thesoluble P content was higher when the initial pH for TCP

solubilization was 8.0 and optimum concentration for TCPsolubilization was 7.5 g l−1.

Keywords Phosphate solubilization .Aspergillusawamori . Phosphate-solubilizing microorganisms.

Introduction

Phosphorus (P) is an essential macronutrient required forplant growth and development (Illmer and Schinner 1992),but unfortunately it is one of the least available and the leastmobile mineral nutrients in the soil (Takahashi and Anwar2007). Deficiency of P in soil is one of the most criticalchemical factors restricting plant growth. As a result, alarge quantity of soluble forms of P fertilizers is appliedevery year to achieve maximum plant productivity. How-ever, the applied soluble forms of P fertilizers are readilyprecipitated into insoluble forms, viz. CaHPO4, Ca3(PO4)2,FePO4 and AlPO4, which again cannot be efficiently takenup by the plants, thus leading to an excess application of Pfertilizers to agriculture land (Omar 1998; Jain et al. 2010).This unmanaged excess of P application causes environ-mental and economic problems.

In India, soil deterioration through accumulation ofexcess salts has attained a serious dimension, especially inthe north-east semi-arid region of Rajasthan where agricul-tural soils are predominantly calcareous and are character-ized by a high pH (pH range 7.0–8.5) and a low amount ofplant available P. The temperature of this region remainsaround 32–45°C in summer and 12–15°C in winter.

Many soil microorganisms act as a rescue by sparinglysolubilizing insoluble inorganic and organic phosphate andproviding it for plant growth (Richardson 2001; Gyaneshwaret al. 2002). Seed or soil inoculation with phosphate-

R. Jain (*) :V. SharmaDepartment of Bioscience and Biotechnology,Banasthali University,Banasthali 304022 Rajasthan, Indiae-mail: rachana_nbs@yahoo.co.in

V. Sharmae-mail: vinaysharma30@yahoo.co.uk

J. SaxenaBiochemical Engineering Department,Kumaon Engineering Collage,Dwarahat,Almora 263653 Uttarakhand, India

J. Saxenae-mail: jyotisaxena2000@yahoo.co.in

Ann Microbiol (2012) 62:725–735DOI 10.1007/s13213-011-0312-8

solubilizing microorganisms (PSMs) is well known toimprove solubilization of fixed soil phosphorus, resultingin higher crop yields (Mittal et al. 2008; Omar 1998).Among all PSMs, fungi are superior to their bacterialcounterpart in phosphate solubilization both on agar and inliquid media (Kucey 1983). The establishment andperformance of these phosphate-solubilizing fungi (PSF)depend on various physiochemical factors such as amountof phosphate, pH, temperature and nature of the insolubleP source. In addition, carbon and nitrogen sources whichparticipate in the active proliferation of fungi andproduction of organic acids strongly influence phosphatesolubilization (Gaur and Sachar 1980; Dave and Patel2003). Since the effective solubilization of phosphate byPSF depends upon the optimum combination of variousphysiochemical factors along with energy sources, thepresent investigation is an effort to evaluate the phosphatesolubilization potential of Aspergillus awamori S19 undervarious physical, nutritional and phosphate sources. Thepresent attempt is to find out the conditions for its futureuse as biofertilizer.

Materials and methods

Fungal isolates

Twelve fungal isolates were obtained from the soilrhizosphere of Pennisetum glaucum (vern. bajra, bajri,pearl millet) in the Krishi Vigyan Kendra Farm, Banasthali,Tonk, in Rajasthan (India). For the collection of therhizosphere soil, 5 plants were uprooted and the looselyadhering soil was removed by mechanical shaking. It wasthen suspended in sterile saline solution and shaken for 6 hat 120 rpm on a rotary shaker. The serial soil dilutions ofthe sample were then individually spread-plated on Pikov-skaya (PVK) agar plates containing 0.5% TCP as insolublephosphate source. The PVK medium having the followingingredients (g l−1distilled water): glucose 10.0; Ca3PO4 2.0;(NH4)2SO4 0.5; yeast extract 0.5; NaCl 0.2; KCl 0.2;MgSO4·7H2O 0.1; MnSO4·H2O 0.002; FeSO4·6H2O 0.002,was sterilized at 121°C pressure for 15 min. pH wasmaintained at 7.00.

After 6 days of incubation at 30°C, the plates wereexamined for the presence of colonies developing clearhalos. Such colonies were picked up, further purified byreplating on agar plates, and maintained on potato dextroseagar slants at 4°C.

Phosphate solubilization in broth medium

Erlenmeyer flasks (250 ml) containing 100 ml of PVKmedium were inoculated with the pure culture of fungal

isolates, incubated at 30°C and 130 rpm on a rotaryincubator shaker for 12 days. The quantitative estimationof soluble P in the culture supernatant was done after every48 h by the molybdenum-blue method (Murphy and Riley1962). The experiment was performed in triplicate. Theamount of phosphate released into culture supernatant wascriteria for choosing the most efficient PSF. The isolateshowing the highest insoluble phosphate-solubilizing activ-ity, named S19, was selected for further study andcharacterized to the species level.

Morphological characterization, ITS1-5.8S-ITS2 regionsequencing and phylogenetic analysis of isolate S19

Prior to molecular characterization, the fungal isolate wasidentified on the basis of cultural and microscopic featuresof the reproductive mycelium, i.e. conidiophore, sporecharacteristics and colony morphology on different media(Czapek yeast extract agar, malt extract agar, 25% glycerolnitrate agar) at different incubation temperature (5, 25, and37°C).

For molecular characterization, DNA was extractedaccording to the procedure described by Lee et al. (1988).The ITS1-5.8S-ITS2 region was amplified by PCR usinguniversal primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATG-3′) (White etal. 1990). PCR reaction was performed in 25 μl as the finalvolume, containing 100–200 ng of DNA, 20 mM Tris-HCl(pH 8.4); 0.1 mM (each) dNTPs; 1.5 mM MgCl2; 0.3 μMof each primer; and 1.5 U of Taq polymerase (BangaloreGenei, India). The reaction mixture was incubated in aMastercycler (BIO-RAD Gene Cycler™) with the follow-ing program: initial denaturation at 95°C for 4.5 minfollowed by 40 cycles of denaturation at 95°C for 30 s,annealing at 52°C for 30 s, and extension phase at 72°C for30 s followed by final extension phase at 72°C for 3 min.

PCR product was separated on 1.2% agarose gel with 1XTAE buffer. Then, the PCR product was purified from theband with the help of Geneaid Biotech Gel/PCR DNAfragment extraction kit. The purified PCR product was sentfor sequencing to Banglore Genei. The ITS1-5.8S-ITS2sequence was deposited in the NCBI under accessionnumber HQ891667 (under processing). The comparativeanalysis of sequences was done using comprehensivedatabase of NCBI (http://www.ncbi.nlm.nih.gov) andnucleotide-nucleotide Basic Local Alignment Search Tool(BLAST) to identify the unknown fungi based on their ITSsequence data.

Effect of temperature and phosphate sources

To study the efficiency of the isolate S19 to solubilizevarious type of phosphate sources, 100 ml of PVK

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containing 0.2% of the tri-calcium phosphate (TCP) wasreplaced with di-calcium phosphate (DCP), Udaipur rockphosphate (URP; P2O5 content=34%, mesh size=74 μm)and ferric phosphate (FP) and inoculated with 1 ml sporesuspension containing 5×105 CFU ml−1. The flasks wereincubated at varying temperatures (i.e. 15, 25, 35 and 45±2°C) on an orbital incubator shaker for 12 days at 130 rpm.The experiment was performed in triplicate. The sampleswere collected after every 48 h. The uninoculated auto-claved medium with different phosphate substrates wasincubated under similar conditions to serve as the control.

Effect of carbon and nitrogen sources

The effect of different carbon and nitrogen sources onphosphate solubilization was observed by replacing glucose(1%) with various carbon sources (fructose, sucrose,maltose, mannitol and sorbitol) and ammonium sulphate(0.5%) with different nitrogen sources [ammonium chloride(AC), ammonium nitrate (AN), sodium nitrate (SN),potassium nitrate (PN) and urea (U)] in PVK medium.Flasks were inoculated with 1 ml spore suspensioncontaining 5×105 CFU ml−1 and incubated at 130 rpm fora period of 6 days at 30±2°C. Uninoculated flasks werekept as control for each set of treatment. The wholeexperiment was conducted in triplicate. The samples werecollected after every 48 h for 6 days.

Effect of initial pH

To find out the optimal initial pH for TCP solubilization byisolate S19, its spores were inoculated into PVK mediumwith initial pH set at 6.0, 7.0, 8.0, 9.0, and 10.0. Flaskswere inoculated with 1 ml spore suspension of 5×105 CFU ml−1 and incubated at 30±2°C and 130 rpm for6 days. Uninoculated flasks were kept as control for eachset of treatment. The whole experiment was conducted intriplicates. The amount of soluble P, pH, and TA of themedium were determined after every 48 h for 6 days.

Effect of TCP concentration

The effect of various TCP concentrations on phosphatesolubilization was studied adding 1, 2, 3, 4, 5, 7.5 and 10 gl−1 to 100 ml PVK medium. Flasks were inoculated andincubated as above. The amount of soluble P, pH, and TA ofthe medium were determined after every 48 h for 8 days.

Chemical analysis of culture broths

The quantitative estimation of soluble P in the culturesupernatant was done after every 48 h by the molybdenum-blue method (Murphy and Riley, 1962). The pH was

recorded using glass electrode pH meter and the titratableacidity (TA) was determined by titrating 5 ml culture filtrateto pH 8.4 with NaOH (Whitelaw et al. 1999).

HPLC analysis of organic acid produced in presence ofdifferent carbon sources was carried out on a SCL-10AVPShimadzu liquid chromatography (Japan) equipped with aSPD-10AVVP Shimadzu UV/Vis detector. The detectorsignal was recorded on Shimadzu Class VP software. Thecolumn used was Whatman ODS-3 column (4.6×150 mm).For organic acid determination, the gradient separation wascarried out using solvent A (90% acetonitrile) and B(0.05% orthophosphoric acid). The nonlinear gradientelution profile was: 0–2 min, solvent B; 2–10 min, gradientfrom 100 to 90% solvent B, maintained at 90% B for15 min. Injection volume was 20 μl and all standards andsamples were injected in triplicate. Additional parametersemployed in HPLC analyses were as follows: columntemperature 30°C, flow rate 1 ml min−1 and detectionwavelength 210 nm. Organic acid standards used were oxalicacid dehydrated (99.5%) and fumaric acid (99.5%) fromFluka BioChemika (Steinheim, Germany) and malic andcitric acids (99%) from Sigma-Aldrich (St. Louis, MO,USA). Quantitation of target analytes in sample wasaccomplished using a multipoint standard calibration curve.

Statistical analysis

All the data are mean of 3 replicates. The data weresubjected to analysis of variance (ANOVA) by using SPSSsoftware, version 16.0 and comparison of means was madeusing Duncan’s multiple range test at p <0.005 levels.

Results

Pennisetum glaucum rhizosphere contained several groupof fungi when plated out on the PVK agar. An isolate coded

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Fig. 1 Phosphate solubilization by Aspergillus awamori S19

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as S19 showed a marked insoluble phosphate solubilizingactivity as visualized by the clarified zone developedaround the colony. In a PVK broth the soluble P showeda gradual increase and reached a concentration of1008 mg l−1 at the 10th day (Fig. 1).

Morphological characterization and phylogenetic analysis

Based on morphological characters, the isolate wasidentified as Aspergillus awamori. The molecular phylo-genetic analysis also placed strain S19 with Aspergillusawamori with 100% identity and 99% query coverage inthe BLAST analysis. Thus, the phylogenetic analysisconfirmed the data obtained from morphological charac-terization of the isolate. The culture was deposited inMicrobial Type Culture Collection (MTCC), IMTECH,Chandigarh, India, and the MTCC number 9630 wasassigned to it.

Phosphate solubilization of different inorganic phosphates(DCP, TCP, URP and FP) at different temperatures

Inoculation of the medium with the test strain significantly(p<0.05) altered the amount of soluble P, pH and TA at allthe temperatures while, uninoculated controls remainedunchanged throughout the experiment except for URP andFP at 45°C. Table 1 clearly shows that significantly highersoluble P i.e. 343 mg l−1 was found at 35°C, during the

12 days experiment that is followed by 326 mg l−1 at 25°Cwith DCP as insoluble P. Three phosphate solubilizationpatterns were observed (Fig. 2A–D); the first patternillustrated continuous but slow rise in soluble P at 15°C,the second pattern which was observed at 25 and 45°Cshowed a peak before decreasing and then a secondincrease, whereas at 35°C the third pattern prevailed whereit reached to a peak and then became constant.

The correlation coefficients (r) at 15°C were −0.88 and0.86 for soluble P versus pH and soluble P versus TA,respectively, when data from the entire incubation periodwere included. In contrast, at 25 and 45°C, soluble P washighly negatively correlated with pH especially for theperiod up to the peak. After this point, the correlationdecreased, with the result that when data from the entireincubation period were analyzed, the correlation coefficientwas found to be much lower. The r values between solubleP and pH at peak were −0.7 and −0.91, whereas it was−0.56 and −0.36 when the data from the entire incubationperiod were included at 25 and 45°C, respectively. At 35°C,the r value for soluble P versus pH before becomingconstant was significant, i.e., −0.92, but when the data fromthe entire incubation period were included, it produced a

Table 1 Solubilization of four different insoluble phosphate sources at various temperatures by A. awamori S19

Phosphatesource

Temperature(°C)

Maximum solubleP (mg l−1)

pH Titratable acidity(mM H+ l−1)

Correlation betweenpH and soluble P

Correlation betweenTA and soluble P

DCP 15 259 d 3.71 d 11.06 b −0.88 0.86

25 326 b 2.72 a 16.74 a −0.56 (−0.70) 0.27 (0.89)

35 343 a 2.90 b 12.7 b −0.44 (−0.92) −0.05 (−0.29)45 322 c 3.44 c 17.81 a −0.36 (−0.91) 0.99

TCP 15 232 c 3.60 b 11.58 b −0.95 0.53

25 261 b 2.52 a 15.54 a −0.96 0.82

35 311 a 4.45 c 14.97 a 0.24 (−0.99) −0.05 (1.00)

45 217 d 5.28 d 5.83 c −0.48 (−0.98) 0.67 (0.61)

URP 15 19 b 4.20 b 3.74 b −0.52 0.87

25 81 a 3.24 a 8.21 a −0.59 (−0.79) 0.64 (0.67)

35 79 a 3.96 a 3.50 c 0.60 −0.5345 Traces NC NC NC NC

FP 15 60 b 3.33 c 10.67 b −0.47 0.99

25 199 a 2.62 a 12.30 a −0.66 (−0.66) 0.72 (0.71)

35 20 c 2.85 b 5.25 c −0.06 0.46

45 Traces NC NC NC NC

Mean values in each column with the same letters do not differ significantly by Duncan’s multiple range test at p≤0.05. Values in parentheses arecorrelation coefficient for the shorter period ,i.e. from day 0 to the day that the P solubilization peak was obtained

DCP di-calcium phosphate, TCP tri-calcium phosphate, URP Udaipur rock phosphate, FP ferric phosphate, NC not calculated

728 Ann Microbiol (2012) 62:725–735

Fig. 2 A–N. Change in soluble P (-ο-), pH (-□-) and TA (-Δ-) overtime during solubilization of DCP, TCP, URP and FP at varioustemperatures by A. awamori S19. DCP di-calcium phosphate, TCP tri-calcium phosphate, URP Udaipur rock phosphate, FP ferric phosphate

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Ann Microbiol (2012) 62:725–735 729

non-significant value. Significantly, the lowest pH wasobserved at 25°C while significantly (p<0.05) highersoluble P was observed at 35°C. Similar results wereobserved for soluble P versus TA.

In the presence of TCP, significantly higher average Psolubilization was observed at 35°C followed by 25°C(Table 1). Similar to DCP, there were 3 phosphatesolubilization patterns as clearly depicted in Fig. 2E–H.The first pattern was found at 15 and 25°C, the secondpattern at 45°C, while the third was at 35°C. The r values

between soluble P and pH at peak and before becomingconstant were−0.98 and −0.99, whereas they were −0.48and −0.24 for data analyzed from the entire incubationperiod, respectively. The r values between soluble P and TAat peak and before achieving constant values were 0.61 and1.0, and when the data from the entire incubation periodwere used for calculation, they were 0.67 and 0.05 for 45and 35°C, respectively.

When ferric phosphate and Udaipur rock phosphatewere used as insoluble P sources, the value of soluble P

I) URP 15oC

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dropped after incubation (Fig. 2I–N). The soluble Pconcentration was found to be lower than the uninoculat-ed control in the early stages of incubation. At 45°C,there was less phosphate solubilization than control flasksand even lesser fungal growth. For URP significantly (p<0.05) highest phosphate solubilization was observed at 35and 25°C, while the optimum temperature for FPsolubilization was 25°C. In the presence of FP and URP,very poor correlation was observed between pH andsoluble P.

Effect of different carbon sources

The effect of various carbon sources on TCP solubilizationrevealed that A. awamori S19 could solubilize insolublephosphate with all carbon sources. In all cases, insolublephosphate solubilization was accompanied by a distinct pHdecrease to pH 2.6–5.7. Amongst them, maltose was bestcarbon source for the phosphate solubilization. The carbonsources in relation to phosphate solubilization activity werein the following order: maltose>glucose>sucrose>fructose>sorbitol>mannitol, showing significant difference amongall. Significantly lowest pH value and highest TA was alsoobserved when maltose was added as carbon source in thegrowth medium (Table 2; Fig. 3). A significant correlationwas observed for soluble P versus pH in all carbon sourcesexcept for maltose.

HPLC analysis of the 6-day-old culture filtrates wasdone to identify and quantify the organic acid producedduring the solubilization of TCP in the presence ofdifferent carbon sources. Oxalic, citric and malic acid(Table 3) were produced in the presence of all 6 differentcarbon sources, whereas succinic acid was produced onlyin the presence of maltose and sucrose. In general, A.awamori S19 produced maximum organic acid in thepresence of sucrose and the minimum in the presence ofmaltose. These results clearly support the correlationstudy between pH and soluble P. In the case of mannitol,organic acid production and phosphate solubilizationwere indirectly proportional to each other.

Effect of nitrogen sources

The effect of different nitrogen sources revealed that all thetested nitrogen sources increased the level of soluble Pproduced. Significantly higher solubilization of phosphoruswas observed in the presence of ammonium chloride (AC)followed by ammonium sulphate (AS) and the lowest wasrecorded with ammonium nitrate (AN) and sodium nitrate(SN). In general, NH4

+ was proved to be better N-sourcethan NO3

−. Significantly lowest pH and highest TA werealso observed in the case of AC (Table 2; Fig. 4). AN andPN did not show any correlation between pH and soluble P

while all other nitrogen sources showed significant corre-lation between pH and soluble P.

Effect of TCP concentration

In general, significant increase in soluble P was recordedwith increase in TCP concentration from 1 to 7.5 g l−1, butno significant increase was observed with 7.5 and 10 g l−1

TCP (Table 2; Fig. 5). Maximum soluble P (556 mg l−1)was recorded at 10 g l−1 TCP concentration on the 4th dayof incubation. Further, the medium receiving lowerquantities of TCP remained more acidic than the mediumreceiving higher amounts but this was not true in the caseof TA which increased with increasing concentration ofTCP.

Effect of initial pH

The change in initial pH of the culture medium influencedphosphate solubilization by the fungus. The solubilizationof TCP was significantly higher in the medium with pH8.00 than that of other acidic (6.00), neutral (7.00) andbasic pH (9.00 and 10.00) (Table 2; Fig. 6). The pH of themedium also decreased significantly on solubilization ofsubstrates, whereas TA increased. Significantly lower pHand higher TA were also observed at initial pH 8.00followed by pH 7.00. At pH 8.00, maximum soluble P innutrient medium was observed at day 4, i.e. 622 mg l−1. Ingeneral, there was a significant correlation coefficientobserved for soluble P versus pH and soluble P and TA atall initial pH except at pH 10.00.

Discussion

Aspergillus awamori S19 was isolated from alkaline andcalcareous rhizosphere soil of Pennisetum glaucum grownin semi-arid environmental conditions. This isolate is likelyto be more successful as a microbial inoculant than themicroorganisms isolated from other soils because of itsability to survive the stress factors that occur in suchregions. There was a significant variation in the quantity ofphosphorus liberated by A. awamori S19 from the differentinorganic phosphates tested (Table 1). The amount ofphosphate solubilized was by far the highest for DCP andTCP among the 4 different phosphate sources. Thesolubilization of URP was also noticeable; however, theperformance was poor in presence of FP. The possiblereason for the poor performance of the A. awamori S19 inFP may be its source of isolation, i.e. alkaline soil wherephosphate is complexed mostly with Ca2+ instead of Fe2+,so no wonder solubilization was higher for the Caphosphate minerals as compared to FP. Moreover, Ca

Ann Microbiol (2012) 62:725–735 731

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Table 2 Effect of different carbon sources, nitrogen sources, TCP concentrations and initial pH of medium on TCP solubilization by A. awamoriS19 in Pikovskaya's broth

Factor Soluble P(mg l−1)

pH Titratable acidity(mM H+ l−1)

Correlation betweenpH and soluble P

Correlation betweenTA and soluble P

Carbon source

Glucose 321 b 3.59 b 9.12 c −0.79* 0.96**

Fructose 245 d 3.78 c 9.00 c −0.93** 0.85**

Maltose 438 a 3.26 a 13.56 a −0.61 0.71*

Sucrose 273 c 3.52 b 7.54 d −0.76* 0.98**

Mannitol 75 f 5.06 e 1.62 e −0.87** 0.68*

Sorbitol 215 e 3.95 d 11.97 b −0.90** 0.91**

Not carbon 24 g 6.45 f 0.61 f −0.44 0.40

Nitrogen source

AN 245 d 4.47 e 6.28 d −0.12 0.70*

AC 413 a 3.49 a 11.88 a −0.75* 0.49

AS 320 b 4.23 d 10.41 b −0.78* 0.89**

PN 268 c 4.15 c 7.06 c −0.30 0.97*

SN 225 d 3.98 b 6.40 cd −0.88** −0.13U 338 b 3.56 a 5.60 d −0.72* 0.84**

TCP concentration

0.1 132 f 3.43 a 6.15 d −0.89** 0.84**

0.2 249 e 3.51 b 8.28 d −0.91** 0.94**

0.3 290 d 3.65 c 9.46 c −0.93** 0.99**

0.4 320 c 4.08 d 8.91 c −0.97** 0.98**

0.5 341 b 4.22 e 10.20 b −0.99** 0.99**

0.75 343 a 4.44 f 10.45 ab −0.98** 0.99**

1.0 359 a 4.47 f 11.28 a −0.94** 0.99**

Initial pH of the medium

6.00 338 b 4.57 c 51.80 a −0.71* 0.99**

7.00 244 d 4.35 b 54.76 a −0.85** 0.47

8.00 416 a 3.71 a 19.76 b −0.85** 0.88**

9.00 300 c 4.73 d 11.12 c −0.79* 0.94**

10.00 16 e 7.72 e 1.07 d NC NC

Mean values in each column with the same letters do not differ significantly by Duncan’s multiple range test at p≤0.05: *p≤0.05, **p≤0.01AN ammonium nitrate, AC ammonium chloride, AS ammonium sulphate, PN potassium nitrate, SN sodium nitrate, U urea, NC not calculated

732 Ann Microbiol (2012) 62:725–735

phosphate minerals showed higher solubility at the pH 3–5which also corresponded to the pH of A. awamori S19(Stumm and Morgan 1995; Whitelaw et al. 1999). Also,according to a few reports, RP, FP and aluminum phosphateare less amenable to microbial solubilization than TCP(Gaur 1990; Seshadri et al. 2004; Shin et al. 2006; Vyas etal. 2007).

Significantly (p<0.05), the optimum temperature forDCP, TCP and URP for P solubilization was found to be35°C, while for FP it was 25°C. A comparatively highertemperature of the soil of the region from where the strainhas been isolated makes it better adapted for highertemperatures, thereby showing the best solubilizationactivity at 35°C. During the study, soluble P concentration,TA and pH often increased and then decreased with time. Apattern of rise and fall in P concentration and TA wasprobably due to organic acid utilization by the fungus underthe last phase of nutrient depletion. Similar observationswere also recorded by Vassilev et al. (1995) and Whitelaw etal. (1999). In general, P solubilization was negativelycorrelated with pH decrease and positively correlated toTA. Below pH 5, the solubility of Ca, Al, Fe (III) and RPminerals in solution increased as pH decreased (Stumm andMorgan 1995; Cerezine et al. 1988). The high negativecorrelation coefficient for soluble P and pH for individual Pminerals at peak provides evidence that in general themechanism of solubilization of phosphate minerals by the

strain S19 might have been the lowering of the pH to a valueat which these compounds were more soluble. Highcorrelation between soluble P and TA observed during thestudy also indicated that acid production was the key Psolubilizing mechanism, as was also reported by Thomas etal. (1985), Nahas (1996), Vassilev et al. (1996) and Whitelawet al. (1999). However, the tendency for the correlationbetween soluble P and pH to become less significant (p<0.05) after the peak indicates that the declinie and then thesecond rise in concentration of soluble P is probably due tofactors other than pH. Hence, it can be inferred that twodifferent mechanisms affecting the increase and decrease insoluble P concentration before and after the peak in Psolubilization were prevailing (Illmer and Schinner 1995).

In the presence of FP and URP, the soluble P concentra-tion was found to be lower than the uninoculated control inthe early stages of incubation. These results predict that thefungus have been consuming more P for supporting its owngrowth and metabolism than it was releasing. The solubili-zation, however, increased at the end of the incubationperiod and would probably have been higher given a longerincubation. This late increase in P solubilization may havebeen due to P liberation after cell lysis or because ofacclimatization to the given environment.

It is well evident from the literature that different fungalspecies/strains prefer different carbon sources. The teststrain was able to solubilize P significantly in the presence

0.00

100.00

200.00

300.00

400.00

500.00

600.00

2 4 6

Incubation time (d)

So

lub

le P

(m

g l-

1 ) AN

AC

AS

PN

SN

U

Fig. 4 Effect of differentnitrogen sources (ANammonium nitrate, ACammonium chloride, ASammonium sulphate, PNpotassium nitrate, SN sodiumnitrate, U urea) on content ofsoluble P

Table 3 Organic acid produced by Aspergillus awamori S19 in the presence of 6 different carbon sources

Carbon source Oxalic acid (g l−1) Malic acid (g l−1) Citric acid (g l−1) Succinic acid (g l−1) Fumaric acid (g l−1)

Glucose 0.97 a 6.95 b 4.29 c - 0.02 a

Fructose 0.23 d 2.68 d 3.61 d - 0.02 a

Maltose 0.69 b 0.56 f 1.14 e 1.84 b -

Sucrose 0.92 a 7.68 a 5.13 b 3.18 a 0.02 a

Mannitol 0.32 c 4.35 c 5.46 a - -

Sorbitol 0.74 b 1.73 e 5.49 a - 0.02 a

Mean values in each column with the same letters do not differ significantly by Duncan’s multiple range test at p≤0.05

Ann Microbiol (2012) 62:725–735 733

of all the tested carbon sources, but maltose was observedas the best carbon source. Aspergillus aculeatus favoredmaltose over glucose, sucrose and mannitol (Gaur andGaind 1983). Barroso et al. (2006) reported that Aspergillusniger solubilized more DCP with maltose and mannitolthan with sucrose in liquid medium. In our case, mannitolwas the least preferred carbon source. Earlier, it has beenreported that sucrose was the preferred carbon source for A.awamori (Gaur and Gaind 1983) and Penicillium rugulo-sum (Reyes et al. 1999). Fungal solubilization of inorganicP is associated with the production of organic acids,most frequently citrate, gluconate, oxalate, and succinate(Cunningham and Kuiack 1992; Gharieb 2000; Vazquez etal. 2000; Reyes et al. 2002; Vassilev et al., 2001). Organicacids may solubilize inorganic P through the release ofacidic protons (Reyes et al. 1999; Whitelaw et al. 1999) andtheir abilities to chelate Ca+2, Fe+3, and Al+3 (Kpomblekouand Tabatabai 1994; Whitelaw et al. 1999). In our study, asignificant correlation was observed for soluble P and pHwith all carbon sources except for maltose. These resultswere also supported by HPLC results. Hence, it can be

further concluded from these results that the main mecha-nism for phosphate solubilization was possibly acidproduction in all carbon sources except in the case ofmaltose. The type of carbon source also affected the type andconcentration of organic acid produced by the fungi which inturn controlled the amount of phosphate solubilization.

In the present study, NH4+ was found to be a good nitrogen

source in comparison to NO3−. The possible reason could be

the acid production in form of H+ release in response to theassimilation of cations such as NH4

+, which has been reportedas a wel- known phenomenon in fungi (Kucey 1983; Roosand Luckener 1984; Asea et al. 1988). The uptake of NH4

+ byfungi in liquid medium commonly led to a rapid drop in pH ofthe medium (Cochrane 1958). Higher acid production andphosphate solubilization from ammonium assimilation hasalso been noted for the solubilization of fluorapatite by A.niger (Cerezine et al. 1988), RP by Penicillium bilaii (Asea etal. 1988), AlPO4 by P. aurantiogriseum and P. simplicissimum(Illmer et al. 1995), TCP by A. aculeatus (Narisan et al. 1994)and CaHPO4 and AlPO4 by P. radicum (Whitelaw et al. 1999).

In the present study optimal pH recorded for phosphatesolubilization was 8.00. The possible reason might be that themicroorganism was isolated from alkaline soil with pHbetween 8.0 and 8.5; hence this fungus is better adapted tothis pH and showedmaximum phosphate solubilization at thispH instead of acidic pH that is optimal for fungal growth.

In conclusion, Aspergillus awamori S19 appears to adaptwell to the stress conditions, and has shown the potential tosolubilize inorganic phosphates under semi-arid conditions.However, there is need for further testing of the fungus inpot and field trials, where conditions are much morecomplex than those prevailing in vitro, before it can berecommended as a bioinoculant.

Acknowledgements Authors are grateful to the vice-chancellor ofthe university for providing the facilities to carry out the work. Theuse of internet and computational facilities of DBT centre ofBioinformatics is truly acknowledged.

References

Asea PEA, Kucey RMN, Stewart JWB (1988) Inorganic phosphatesolubilization by two Penicillium species in solution culture andsoil. Soil Biol Biochem 20:459–464

Barroso CB, Pereira GT, Nahas E (2006) Solubilization of CaHPO4 andAl PO4 by Aspergillus niger in culture media with different carbonand nitrogen sources. Braz J Microbiol 37:434–438

Cerezine PC, Nahas E, Banzatto DA (1988) Soluble phosphateaccumulation by Aspergillus niger from fluoroapatite. ApplMicrobiol Biotechnol 29:501–505

Cochrane VW (1958) Physiology of fungi. Wiley, New YorkCunningham J, Kuiack C (1992) Production of citric and oxalic acids

and solubilization of calcium phosphate by Penicillium bilaii.Appl Environ Microbiol 58:1451–1458

TCP concentration (g l-1)0 2 4 6 8 10 12

So

lub

le P

(m

g l-1

)

100

150

200

250

300

350

400

Fig. 6 Effect of various TCP concentrations on amount of soluble P

Initial pH5 6 7 8 9 10 11

So

lub

le P

(m

g l-1

)

0

100

200

300

400

500

Fig. 5 Effect of initial pH on amount of soluble P

734 Ann Microbiol (2012) 62:725–735

Dave A, Patel HH (2003) Impact of different carbon and nitrogensources on phosphate solubilization by Pseudomonas fluorescens.Indian J Microbiol 43:33–36

Gaur A (1990) Phosphate solubilizing microorganisms as biofertil-izers. Omega, New Delhi, India, pp 40–44

Gaur AC, Gaind S (1983) Microbial solubilization of phosphate withparticular reference to iron and aluminum phosphate. Sci Cult 49:110

Gaur AC, Sachar S (1980) Effect of rock phosphate and glucoseconcentration on phosphate solubilization by Aspergillus awa-mori. Curr Sci 49:553–554

Gharieb MM (2000) Nutritional effects on oxalic acid production andsolubilization of gypsum by Aspergillus niger. Mycol Res104:550–556

Gyaneshwar P, Kumar GN, Parekh LJ, Poole PS (2002) Role ofmicroorganisms in improving P nutrient of plants. Plant Soil245:83–93

Illmer P, Schinner F (1992) Solubilization of inorganic phosphate bymicroorganism isolated from forest soil. Soil Biol Biochem24:389–395

Illmer P, Schinner S (1995) Solubilization of inorganic calciumphosphates- Solubilization mechanisms. Soil Biol Biochem27:257–263

Illmer P, Barbato A, Schinner F (1995) Solubilization of hardly-soluble AlPO4 with P-solubilizing microorganisms. Soil BiolBiochem 27:265–270

Jain R, Saxena J, Sharma V (2010) The evaluation of free andencapsulated A. awamori for phosphate solubilization in fermen-tation and soil-plant system. Appl Soil Ecol 46:90–94

Kpomblekou K, Tabatabai MA (1994) Effect of organic acids onrelease of phosphorus from phosphate rocks. Commun Soil SciPlant Anal 22:63–73

Kucey RMN (1983) Phosphate solubilizing bacteria and fungi in variouscultivated and virgin Alberta soils. Can J Soil Sci 63:671–678

Lee SB, Milgroom MG, Taylor JW (1988) A rapid high yield mini-prep method for isolation of total genomic DNA from fungi.Fungal Genet News 35:23–24

Mittal V, Singh O, Nayyar H, Kaur J, Tewari R (2008) Stimulatoryeffect of phosphate-solubilzing fungal strain (Aspergillus awa-mori and Penicillium citrinum) on the yield of chickpea (Cicerarietinum L. Cv. GPF2). Soil Biol Biochem 40:718–727

Murphy J, Riley HP (1962) A modified single solution method for thedetermination of phosphate in natural waters. Anal Chim Acta27:31–36

Nahas E (1996) Factor determining rock phosphate solubilization bymicroorganism isolated from soil. World J Microbiol Biotechnol12:18–23

Narisan V, Thakkar J, Patel HH (1994) Isolation and screening ofphosphate solubilizing fungi. Indian J Microbiol 34:113–118

Omar SA (1998) The role of rock phosphate solubilizing fungi andvesicular arbuscular mycorrhiza (VAM) in growth of wheat plantsfertilized with rock phosphate. World J Microbiol Biotechnol14:211–219

Reyes I, Bernier L, Simard R, Tanguay PH, Antoun H (1999)Characteristics of phosphate solubilization by an isolate of a

tropical Penicillium rugulosum and two UV induced mutants.FEMS Microbial Ecol 28:291–295

Reyes I, Bernier L, Antoun H (2002) Rock phosphate solubilizationand colonization of maize rhizosphere by wild and geneticallymodified strains of Penicillum rugulosum. Microbial Ecol 44:39–48

Richardson AE (2001) Prospects for using soil microorganisms toimprove the acquisition of phosphorus by plants. Aust J PlantPhysiol 28:897–906

Roos W, Luckener M (1984) Relationships between proton extrusionand fluxes of ammonium ions and organic acids in Penicilliumcyclopium. J Gen Microbiol 130:1007–1014

Seshadri S, Ignacimuthu S, Lakshminarasimhan C (2004) Effect ofnitrogen and carbon sources on the inorganic phosphatesolubilization by different Aspergillus niger strain. Chem EngCommun 191:1043–1052

Shin W, Ryu J, Kim Y, Yang J, Madhaiyan M, Sa T (2006) Phosphatesolubilization and growth promotion of maize (Zea mays L.) bythe rhizosphere soil fungus Penicillium oxalicum. 18th WorldConference of Soil Science, 9–15 July, Philadelphia

Stumm W, Morgan JJ (1995) Aquatic chemistry. Chemical eqilibriaand rates in natural waters, 3rd edn. Wiley, New York

Takahashi S, Anwar MR (2007) Wheat grain yield, phosphorusuptake and soil phosphorus fraction after 23 years of annualfertilizer application to an Andosol. Field Crops Res 101:160–171

Thomas GU, Shantaram MV, Saraswathy N (1985) Occurrence andactivity of phosphate-solubilizing fungi from coconut plantationsoils. Plant Soil 87:357–364

Vassilev N, Baca MT, Vassileva M, Franco I, Azcon R (1995) Rockphosphatesolubilization by Aspergillus niger grown on sugar-beetwaste medium. Appl Microbiol Biotechnol 44:546–549

Vassilev N, Franco I, Vassileva M, Azcon R (1996) Improved plantgrowth with rock phosphate solubilized by Aspergillus nigergrown on sugar-beet waste. Biores Technol 55:237–241

Vassilev N, Vassileva M, Fenice M, Federici F (2001) Immobilizedcell technology applied in solubilization of insoluble inorganic(rock) phosphates and P plant acquisition. Biores Technol79:263–271

Vazquez P, Holguin G, Puente M, Lopez-Cortes A, Bashan Y (2000)Phosphate solubilizing microorganisms associated with therhizosphere of mangroves in a semi arid coastal lagoon. BiolFert Soils 30:460–468

Vyas P, Rahi P, Chauhan A, Gulati A (2007) Phosphate solubilizationpotential and stress tolerance of Eupenicillium parvum from teasoil. Mycol Res 3:931–936

White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and directsequencing of fungal ribosomal RNA genes for phylogenetics.In: Innis MA, Gellfand DH, Sninisky JJ, White TH (eds) PCRprotocol: aguide to methods and applications). Academic, NewYork, pp 315–322

Whitelaw MA, Harden TJ, Helyar KR (1999) Phosphate solubilizationin solution culture by the soil fungus Penicillium radicum. SoilBiol Biochem 32:655–665

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