Phosphate solubilization in vitro by isolatedAspergillus niger and Aspergillus carbonarius

Chunqiao Xiao • Yujuan Fang • Ruan Chi

Received: 25 March 2013 / Accepted: 10 September 2013

� Springer Science+Business Media Dordrecht 2013

Abstract Two strains of Aspergillus, A. niger and A. carbonarius, were isolated

from agricultural soil and a lake, respectively, in China. The two isolates could

effectively release soluble phosphate in NBRIP medium containing Ca3(PO4)2 as

the sole phosphorus source. Acidification of the broth seemed to be the major

mechanism for phosphate solubilization by the isolates, and this had a significant

correlation with a drop in pH. High-pressure liquid chromatography analysis indi-

cated the participation of various organic acids, with gluconic acid as the principal

component in the process of acidification in the broth. The isolates displayed trends

of soluble phosphate release that closely matched the substantial increases in glu-

conic acid concentration. Abiotic incubation study using organic or inorganic acid

to solubilize Ca3(PO4)2 indicated that the content of soluble phosphate released was

significantly lower than that of the broth inoculated with the isolates. Higher release

of soluble phosphate and pH reduction have occurred when ammonium rather than

nitrate served as the sole source of nitrogen with Ca3(PO4)2.

Keywords Phosphate solubilization � Aspergillus niger � Aspergillus

carbonarius � Soluble phosphate � Ca3(PO4)2

Introduction

Phosphorus (P) plays a vital role in plant nutrition. P can be tightly bound with soil

cations, particularly calcium, iron, or aluminum, leading to precipitation of P in the

soil. Therefore, despite P being widely and abundantly distributed in the soil in both

C. Xiao � Y. Fang � R. Chi (&)

Key Laboratory for Green Chemical Process of Ministry of Education, Wuhan Institute of

Technology, Wuhan 430073, People’s Republic of China

e-mail: whjhx@aliyun.com

123

Res Chem Intermed

DOI 10.1007/s11164-013-1395-6

its inorganic and organic forms, it is not readily soluble in the soil and not easily

accessible for plant growth.

Phosphate solubilization by fungi is widespread and, with respect to agriculture,

considerable attention has been paid to this process [1]. The advantage of using

fungi for such a process includes their tolerance to potentially toxic metals, and

better acid and alkali tolerance than bacteria, although fungi might be inferior to

bacteria in their ability to colonize plant roots. Several studies have shown that an

increase in yield or plant growth can be achieved through the inoculation of

phosphate-solubilizing fungi in either pot experiments or under field conditions [2–

4]. Therefore, fungi may have a much better potential to serve as an agent to convert

insoluble phosphates into a soluble form (e.g., HPO42-, H2PO4

-) usable by plants in

the soil.

Fungi are used widely in biotechnology for many processes, including production

of organic acids, antibiotics, enzymes, food products, and alcohol, etc. Organic

acids production by fungi has been reported to be the main mechanism for the

solubilization of inorganic phosphates [5, 6]. These organic acids can either dissolve

inorganic phosphates as a result of anion exchange or can chelate Ca, Fe or Al ions

associated with these inorganic phosphates [7]. However, incongruent observations

have also been reported. Illmer and Schinner [8] reported that, when growing a

phosphate-solubilizing Penicillium sp. in an inorganic phosphates amended

medium, none of the 24 organic acids assayed were produced in significant

amounts. Chen et al. [9] also reported that some bacterial isolates showing

capacities of phosphate solubilization did not produce any kinds of organic acids.

Some reports showed that proton excretion accompanied NH4? uptake was the

possible mechanism causing phosphate solubilization by fungi [10, 11]. However,

there were also some inconsistent reports [12]. Actually, there have been many

different observations about the mechanisms for phosphate solubilization by fungi,

and they should be studied in depth since there is current interest in the use of fungi

capable of solubilizing inorganic phosphates.

Filamentous fungi, especially genus Aspergillus, are known to secrete certain

organic acids as a part of their metabolic activity, and the solubilization of different

types of inorganic phosphates by them has already been demonstrated [13–15].

Although many fungi can be used to produce organic acids, the genus Aspergillus

remains the main industrial producer [16]. In this study, two species of Aspergillus,

namely A. niger and A. carbonarius, were isolated from agriculture soil and a lake

in China, respectively, and their ability to solubilize Ca3(PO4)2 was studied. The

characteristics and mechanisms for phosphate solubilization by the two isolates

were also investigated.

Materials and methods

Sampling and isolation of probable phosphate-solubilizing fungi

Strain WHAT2 (A. niger) was isolated from soil samples from the rhizosphere of

wheat in a farm located in the suburb of Wuhan city. Strain WHAS3 (A.

C. Xiao et al.

123

carbonarius) originated from water samples from the east lake of Wuhan City.

Isolation and purification were carried out as follows: 10-g or 10-ml samples each

were added to 100-ml sterile saline (0.5 % NaCl), and mixed on the magnetic

blender for 20 min to completely separate microorganisms from the samples. The

serially diluted sample solutions were spread on modified National Botanical

Research Institute’s phosphate growth (NBRIP) agar (pH 7), which contained (per

liter): 5 g Ca3(PO4)2, 10 g glucose, 0.5 g (NH4)2SO4, 0.2 g KCl, 0.5 g

MgCl2�6H2O, 0.25 g MgSO4�7H2O, and 20 g agar [17]. The isolates were incubated

at 30 �C for 3–5 days until the colonies appeared. A single colony with a clear zone

around the fungal colonies from different sites was then picked out for the next

inoculation. Among these fungi, A. niger and A. carbonarius were selected. The

transfer was repeated until the pure culture was obtained. Pure cultures were

maintained on potato dextrose agar slants and kept at 4 �C until required for further

studies. Their identities were confirmed based on the gene sequencing of the internal

transcribed spacer (ITS) regions of the ribosomal DNA (rDNA). The ITS 1, 5.8S

rRNA gene and ITS 2 were amplified by using the polymerase chain reaction

according to White and colleagues. [18]. Amplified products were sequenced and

analyzed using the BLAST searching program at the National Center for

Biotechnology Information website: http://www.ncbi.nlm.nih.gov/BLAST/. Rela-

ted sequences were preliminarily aligned with the default setting of Clustal X (2.0)

[19]. Phylogenetic and molecular evolutionary analyses were conducted using

MEGA v.5 [20].

Broth assays for phosphate solubilization by the isolates

Broth assays were carried out in shake flasks with 50 ml NBRIP medium containing

0.5 g Ca3(PO4)2 as sole P source. The initial pH of the medium was adjusted to 7.

Mycelial discs (10 mm) of each isolate from actively growing colonies after 4 days

on modified NBRIP agar were added as inoculum. Flasks were shaken at 160 rpm at

30 �C for 10 days. Autoclaved, uninoculated medium served as control. Samples

from flasks were taken every other day and the broth was centrifuged at 11,000g for

20 min, and the supernatant was filtered. The filtrate was then assessed for the

soluble phosphate, pH, and organic acids. All experiments were performed in

triplicate.

Abiotic phosphate solubilization by organic and inorganic acids

To study the importance of organic acid in phosphate solubilization, gluconic acid

and acetic acid were added to the above medium containing Ca3(PO4)2 as sole P

source to give a concentration similar to that of the gluconic acid detected in the

broth inoculated with the isolates. To study the importance of acidity in phosphate

solubilization, in another set of P-amended medium, 0.1 M HCl and H3PO4 were

added until the pH was as close as possible to that of the broth inoculated with the

isolates. Each flask was inoculated and incubated at room temperature for 2 h prior

to subsampling. The solution was centrifuged and assessed for the content of soluble

phosphate.

Phosphate solubilization in vitro by isolated A. niger and A. carbonarius

123

Effect of nitrogen sources on phosphate solubilization by the isolates

The effect of nitrogen sources on phosphate solubilization by the isolates was

evaluated by replacement of ammonium sulfate with five nitrogen sources, viz.

ammonium chloride, ammonium nitrate, potassium nitrate, sodium nitrate, and

calcium nitrate, respectively. Culture supernatant was sampled from each broth for

the assessment of the soluble phosphate and pH.

Analytical methods

The content of soluble phosphate was determined by using the vanadium–

ammonium molybdate colorimetric method with a UV–Vis 8500 spectrophotometer

at 490 nm [21]. The pH was recorded with a pH meter equipped with a glass

electrode. Organic acids in the broth were determined by high-pressure liquid

chromatography (HPLC; Agilent 1100) analysis using C18 columns (Thermo

Electron) [22]. Organic acids standards included citric, oxalic, gluconic, formic, a-

ketoglutaric, fumaric, lactic, pyruvic, succinic, tartaric, and ascorbic acids.

Phosphatases activity was determined using the method described by Tabatabai

and Bremmer [23]. Values were given as mean ± standard deviation for triplicate

samples.

Results and discussion

Identification of the isolates

The two isolates were filamentous fungi that produce microscopic spores inside

sacs. They can grow in wide ranges of temperature and pH at 10–45 �C and 2.0–9.5,

and the best temperature and pH are at 28–30 �C and 5.5–6.5, respectively. The

isolates were finally identified as A. niger and A. carbonarious based upon the

results of phenotypic characterization and the phylogenetic tree constructed on the

basis of ITS sequence data (Fig. 1). The sequences were deposited in the GenBank

nucleotide sequence data library under the following accession numbers: JQ929762

(WHAT2) and JQ929763 (WHAS3), respectively.

Phosphate solubilization by the isolates

The two isolates, namely A. niger WHAT2 and A. carbonarius WHAS3, could

effectively solubilize Ca3(PO4)2 in NBRIP medium compared to the abiotic control,

and the content of soluble phosphate released increased significantly during 10 days

of solubilization of Ca3(PO4)2, although there was a slight decrease after 6 days

(Fig. 2). The two isolates varied in their capacities to release soluble phosphate from

Ca3(PO4)2, and the solubilization of Ca3(PO4)2 by strain WHAT2 was better than

that by strain WHAS3 in this study.

Figure 3 shows that the pH in the broth decreased sharply after inoculation

compared to the abiotic control, and remained almost constant after 6 days. A. niger

C. Xiao et al.

123

WHAT2 effected the larger reduction of pH than A. carbonarius WHAS3. The

release of soluble phosphate in the broth was associated with a concomitant

decrease in pH. Simple regression analysis suggest that there is a significant

negative correlation (r = -0.76; P \ 0.01) between the soluble phosphate released

and pH. This observation is consistent with previous reports demonstrating that

phosphate solubilization with decrease in pH [24].

Various organic acids, including gluconic, oxalic, formic, citric, pyruvic,

succinic, and lactic acid, were detected in the broth during solubilization of

Ca3(PO4)2 by HPLC analysis (Table 1). It is presumed that the production of

organic acids plays a vital role in the acidification of the broth. There was a

significant decrease of pH in the broth facilitating the solubilization of Ca3(PO4)2.

gi 161408454 dbj AB369898.1 Aspergillus niger

WHAT2

gi 307088982 gb HM801882.1 Aspergillus sp.

WHAS3

gi 349844858 gb JF Aspergillus carbonarius

gi 158144421 gb EF661429 Aspergillus ochraceopetaliformis

gi 158144422 gb EF661430 Aspergillus insulicola

gi 158138942 gb EU02161

838359.1

.1

.1

6.1 Aspergillus flocculosus41

100

99

99

88

Fig. 1 ITS rDNA-based phylogenetic relationship between the isolates and representatives of otherrelated taxa (GenBank accession numbers in parentheses). The numbers at the nodes indicate the levels ofbootstrap support based on data for 1,000 replicates; values inferred greater than 50 % are only presented.Scale bar 0.01 substitutions per nucleotide position

0 2 4 6 8 100

50

100

150

200

250

300

Con

tent

of s

olub

le p

hosp

hate

(m

g l-1

)

Time (d)

Abiotic control WHAT2 WHAS3

Fig. 2 Content of soluble phosphate in the broth inoculated with the isolates during 10 days ofsolubilization of Ca3(PO4)2. Bars standard errors

Phosphate solubilization in vitro by isolated A. niger and A. carbonarius

123

Among the organic acids detected, gluconic acid was predominantly produced by

the two isolates in this study. The results in Fig. 4 show that the concentration of

gluconic acid in the broth inoculated with the isolates increased significantly during

10 days of solubilization of Ca3(PO4)2, although there was a slight reduction after

6 days. A. niger WHAT2 produced the highest concentration of gluconic acid

compared with A. carbonarius WHAS3 during solubilization of Ca3(PO4)2.

The two isolates displayed trends of release of soluble phosphate that closely

matched the substantial increases in gluconic acid concentration. Simple regression

analysis shows that there is a strong positive correlation between the content of

soluble phosphate released and the concentration of gluconic acid (r = 0.86;

P \ 0.01).

Abiotic phosphate solubilization by organic and inorganic acids

The production of organic acids, especially gluconic acid, played a vital role in the

solubilization of Ca3(PO4)2 in the present study. This result prompted us to

investigate the effect of organic and inorganic acids on the solubilization of

Ca3(PO4)2 under sterile conditions. In the present study, the importance of organic

acid in the solubilization of Ca3(PO4)2 was assessed by adding gluconic acid and

acetic acid to the medium containing Ca3(PO4)2, at a concentration similar to the

level of gluconic acid detected in the medium inoculated with the two isolates.

However, this strategy failed to affect the release of soluble phosphate in the broth

to the comparable levels in the presence of the isolates. As shown in Table 2, it was

obvious that the organic acid solubilized less Ca3(PO4)2 than by A. niger WHAT2.

Similar results were observed in the presence of A. carbonarius WHAS3 (data not

0 2 4 6 8 103.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

pH

Time (d)

Abiotic control WHAT2 WHAS3

Fig. 3 pH in the broth inoculated with the isolates during 10 days of solubilization of Ca3(PO4)2. Barsstandard errors

C. Xiao et al.

123

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Res

ult

sre

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sen

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ot

det

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d

Phosphate solubilization in vitro by isolated A. niger and A. carbonarius

123

shown). The results confirmed some reports demonstrating that the mere presence of

organic acids does not account for all the soluble phosphate that is solubilized by

microorganisms [25].

Likewise, the effect of inorganic acid (HCl and H3PO4) on the solubilization of

Ca3(PO4)2 under sterile conditions was also studied. When the pH of the medium

was finally adjusted to different pH values similar to the level detected in the

medium inoculated with A. niger WHAT2, only a small amount of soluble

phosphate was released, which was significantly smaller than the content detected in

the medium incubated with the test fungus (Table 2). Similar results were observed

in the presence of A. carbonarius WHAS3 (data not shown). Our results further

demonstrated multiple actions in addition to acid production of phosphate-

solubilizing microorganisms in the phosphate solubilization.

Effect of nitrogen sources on phosphate solubilization by the isolates

Better phosphate solubilization and pH reduction were observed when NH4? rather

than NO3- was used as the nitrogen source (Table 3). The results further confirmed

that H? extrusion by NH4? assimilation may also be involved in the solubilization

of inorganic phosphates. However, the hypothesis that phosphate solubilization is

linked to acidification caused by NH4? assimilation does not hold true for all

microorganisms [12, 26].

Phosphate-solubilizing microorganisms are known to produce phosphatases,

which are hydrolytic enzymes, responsible for the breakdown of insoluble

phosphates. Achal et al. [27] reported that phosphate solubilization by Aspergillus

tubingensis is due to the lowering of the pH of the broth and the activity of the acid

phosphatase and phytase. Rinu and Pandey [28] have also shown that phosphatases

0 2 4 6 8 100

100

200

300

400

500

Con

cent

ratio

n of

glu

coni

c ac

id (

mg

l-1)

Time (d)

WHAT2 WHAS3

Fig. 4 Concentration of gluconic acid in the broth inoculated with the isolates during 10 days ofsolubilization of Ca3(PO4)2. Bars standard errors

C. Xiao et al.

123

Ta

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Day

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Con

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trat

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ph

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(mg

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Co

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n

(mg

l-1)

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e

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ased

(mg

l-1)

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ion

(mg

l-1)

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ased

(mg

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osp

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(mg

l-1)

pH

So

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ph

osp

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ased

(mg

l-1)

23

80

.7±

18

.74

.35

±0

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12

2.7

±5

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80

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4.3

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2.8

±0

.64

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±0

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44

49

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25

.34

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44

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44

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7.7

±0

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96

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0.4

64

68

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24

.53

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±0

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27

2.8

±1

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46

8.5

10

1.2

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.84

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3.9

13

1.2

±1

.13

.91

7.7

±0

.5

84

41

.8±

20

.64

.07

±0

.14

26

3.7

±1

3.4

44

1.8

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.3±

5.5

44

1.8

7.0

±0

.34

.07

30

.1±

0.7

4.0

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.4±

0.3

10

42

8.7

±2

1.8

4.1

4±

0.2

02

55

.9±

12

.14

28

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±3

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4.1

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±0

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Res

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sre

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Phosphate solubilization in vitro by isolated A. niger and A. carbonarius

123

Ta

ble

3E

ffec

to

fam

mo

niu

man

dn

itra

teo

nth

ere

leas

eo

fso

lub

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ate

fro

mC

a 3(P

O4) 2

by

the

isola

tes

afte

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day

sin

ocu

lati

on

(NH

4) 2

SO

4N

H4C

lN

H4N

O3

Ca(

NO

3) 2

KN

O3

NaN

O3

WH

AT

22

83

.7±

11

.9

(4.1

9±

0.0

7)

27

1.6

±1

0.0

(4.2

4±

0.1

0)

24

3.4

±1

1.2

(4.3

7±

0.2

5)

20

1.3

±1

2.4

(4.4

8±

0.1

0)

20

1.3

±1

0.5

(4.4

7±

0.0

8)

20

2.8

±1

2.4

(4.4

5±

0.1

3)

WH

AS

32

47

.2±

10

.8

(4.5

0±

0.0

9)

24

1.5

±9

.3

(4.6

2±

0.1

3)

22

1.5

±1

1.7

(4.7

0±

0.1

7)

15

8.7

±9

.7

(4.7

7±

0.0

9)

17

8.5

±1

0.7

(4.7

8±

0.0

8)

16

2.3

±8

.3

(4.7

7±

0.1

1)

Res

ult

sre

pre

sen

tth

em

ean

of

thre

ere

pli

cate

s±

stan

dar

dd

evia

tio

n.

Fig

ure

sin

par

enth

eses

indic

ates

pH

of

the

bro

thaf

ter

6d

ays

ino

cula

tio

n

C. Xiao et al.

123

excreted by Paecilomyces hepiali were involved in the solubilization of insoluble

phosphates. However, we failed to detect any phosphatases in the broth in the

present study.

Generally, phosphate solubilization by microorganisms is not a simple phenom-

enon and may be determined by many factors, such as nutritional, physiological,

and the growth conditions of the microorganisms. Therefore, it could be

accomplished by a range of mechanisms and need further study.

Conclusion

Two isolated strains, namely A. niger WHAT2 and A. carbonarius WHAS3, can

effectively solubilize Ca3(PO4)2 and release soluble phosphate in NBRIP medium.

The main mechanism of phosphate solubilization is the production of organic acids

(principally gluconic acid), which played a vital role in the acidification of the broth,

followed by the decrease in pH, and thus further facilitated the solubilization of

Ca3(PO4)2. An abiotic study using organic or inorganic acid to solubilize Ca3(PO4)2

indicated that the content of soluble phosphate released was significantly lower than

was detected in the broth inoculated with the isolates. The enhancement of the

release of soluble phosphate and pH reduction was observed when ammonium

rather than nitrate acts as the sole source of nitrogen with Ca3(PO4)2 in NBRIP

medium.

Acknowledgments This research work was kindly supported by National Natural Science Foundation

of China (51004078), Program for New Century Excellent Talents in University (NCET-11-0965),

National Natural Science Foundation of Hubei province (2012FFA101), Program for Changjiang

Scholars and Innovative Research Team in University (No. IRT0974) and National Basic Research

Program of China (No. 2011CB411901).

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