Solubilization of insoluble phospates by Aspergillus niger

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/

ISSN 0215 - 9318

JURNAL PENELITIAN BIOTEKNOLOGI PERKEBUNAN

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Menara Perkebunan. 2000. 68(2) 37 47

Solubilization of insoluble phosphates by

Aspergillus niger

Pelarutanfosfat sukar larUE oleh Aspergillus niger LAKSMITA P. SANTII), D. H. GOENADrI), SISWANTOi),

I. SAILAI-e) & ISR0I3 )

I) Biotechnelogy Research Unit for Estate Crops, Bogor 1MSI, Indonesia

2) Bogor Agricultural University, Bogor, Indonp.sia 3) Graduate School, Boger Agricultural University, Bogor, Indonesia

Ringkasan

Penggunaan /angsung fosfat a/am (FA) ke da/am tanah sebagai sumber pupuk P te/ah dila-kukan se/ama bertahun-tahun melalui beberapa macam cara penggunaan. Kualitas FA di Indo-nesia umumnya rendah dan ketersediaan bahan baku yang berkualitas untuk produksi pupllk fosfat ter/arut relatif terbatas. Beberapa mikroba asal tanah yang dapat melarutkan fosfat anorganik lelah banyak dilaporkan. Nanwl1, informasi yang tersedia ten tang mekanisme peiarlltan P dari FA lokal asal Indonesia don P anorganik o/ell Aspergillus niger BCC FI94 belum banyak dite/iti. Satu seri penelitia."l laboralorium lelah dilaksana-kan untuk mengetahlli kelllamplian A. niger BCC FI94 melarutkan P. Evaillasi agronomi FA lokal (FA Cilellngsi dan Madura) di rllmah kaea juga telah diiakukan. A. niger BCC F 194 dapal melarutkan sumber P Sltkar larut, yailu FA Cileungsi dan Madura. serta senyawa CaJ(PO.J2 dan AIP04. Kelarutan P anorganik lersebut ber-hubungan dengan peningkatan aktivitas proton (ff) yang menyebabkan penurunan pH medium dan produksi asam organik. Asam organik ulama yang dihasilkan oleh A. niger BCC FI94 dalam medium cair Pikovskaya yang dimodiflkasi adalah asam oksa/at (3.75 mM). asam silral (2.0 mM). dan asam glukonat (O.Q mM). Kelarulan FA CilelIngsi I"bih besar dibandingkan dengan FA Madura. dan kelarulan CalPO)2 lebih besar dibandingkan kelarlltan AIP04• Tidak ada korelasi anlara kdarutan P anorganik dengan aktivitas enzim fosfatase. walaupun aktivitas enzim fosfa-lase cukup linggi terdeteksi dalam medium. Salu formula biosuperfosfat lelah berhasil dirakit

dengan mereaksikan FA lokal dengan supernatan kultur eair (SKC) pengganti Qsam sulfat. Hasil percobaan pada bibit kakao. kpret dan kelapa sawil di rumah kaea menunjukkan bahwa proto-lipe pupuk biosuperfosfa! dengan bahan baku FA Cileungsi dan Madura bentllk granul ma/lplln serbllk. memiliki ni/ai keefektifan agronomi yang relatif menyamai SP-konvensional.

Summary

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SantI ct al.

decrcase and production of organic acid. The

major acidic mctabolites produced by A. niger

BCC F 194 in modified liquid Pikovskaya medium were oxalic acid (3.75 mM), citric acid (2.0 mM), and gluconic acid (0.9 mM). The solubi!ization of

Cileungsi RP was higher than that of Madura RP,

and the solubilization of C"3(P04h \\'flS better than

that of AIP04 . There is no correlation between

solubilization of inorganic

P

and ･ｮＮｾｹｭ･＠

activities, although high level of activity of

phosphatase enzyme \Vas detectaJ!e in the

medium. A biosuperphosphate formula had heen

constructed by reac.ting local RP with liquid

culture supernatant (LCS) rcplacing sulfuric acid. In the green housc experiments using cocoa, rubber, and oil palm seedlings, both granular and powder biosuperphosphate prototypes showed a comparable relative agronomic effectiveness value to that of the conventional SP.

[Keywords: P solubilization, Aspergillus niger,

rock phosphate]

Introduction

Most rock phosphate deposits found in the world is classified as low reactive RPs and therefore, it cannot be used successfully as phosphorus (P) sources for crop produc-tion. The composition of these rock phos-phates varies from one deposit to another. Most of the world rock phosphates are-of sedimentary origin. Large rock phosphate

deposits estimated 1-2 million tonnes, are

distributed in different pans of Indonesia

(Moersidi, 1999). Since these deposits are

low in phosphorous content «25% P20 S) and

contain some impurities like oxides of Si, Fe, AI, and Ca, they are unsuitable for the

manufacture of superphosphate (Narsian el

al., 1993). The conventional method for en-hancing the rock phosphate availability is to increase its solubility by treating with inor-ganic acids, mainly sulphuric acid and phos-phoric acid but, this approach is not appli-cable because of high capital production

(Hammond et al., 1989, Goenadi el al.,

2000). A very attractive approach for rock phosphate solubilization is the appiication of microbes capable of excreting organic acids

(Gerke, 1992).

Many soil microbes are known to solu-biliL:e insoluble forms of inorganic phosphatic

compounds. [n vitrc studies with microbial

isolates from ｾｯｩｬ＠ indicated that fungi were

more efficient in thc solubilization of

inor-ganic phosphate as compared 10 bacteria

(Thomas et ai., \985; Nahas 1996; Goenadi

el 01., 199LJ). Filamentous fungi are widdy

used as producers of organic acid:;, particular-ly A. niger and some Penicillium sp., which

have been tested i!1 fermentation systems or

inoculated directly into soil in order to

solu-bilize rock phosphate (Vassilev et al.. \997;

V asi leva et al., 1998).

Acidification of soil is a consequence of natural and anthropogenic processes (Ulrich

& Sumner, 1991). One of the outstanding changes in soil due to acidification is the

mobilization of Ae+ ions, which are toxic to

plants on one hand and cause chemical fixa-tion of plant-available P on the other hand. Hardly-soluble aluminum phosphates are for-med and become the largest P-fraction in

many acidic soils (McLean, 1976). AIP04• i:1

the contrary to calsiui7l phosphate, will never play an important role il1 ameliorating soils. Leaving that out of consideration, the

impor-tance of Alr04 solubilization fOf soil

forma-tion, mineral transformation and AI-toxicity

is obvious (lllmer et al., 1995). In this

inves-tigation, pure insoluble inorganic phosrhates

i. e. aluminum phosphate (A IP04 ) and ca

1-cium phosphate (CaJ(P0₄₎₂₎have been used

for expressing phosphate :,olubi!izillg activity ofA. niger BeC f194.

Natural RPs has been recognized as a valuable alternative source for P fertilizer, especially for acid soils. The economic value of the rocks increases considerably along with the increasing costs of SP production

(Goenadi et al., 2000). Consequently, there is

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Soiubilizatiol: if insoluble phosphates by Aspergillus niger

a growing interest in ways of manipulating such rock to obtaIn a more valuable product,

i.e. partially acidulating RPs (Goenadi, 1990; Rajan & Ghani, 1997), reacting with

syn-thetic organic acids (Sagoe et al., 1998) or

natural organic acids (Singh & Amberger,

1998), and decrea:;ing particle size (Babare et

aI,

1997). Goenadi et al. (2enO) repofled that

Liquid Culture Supernatant (LCS) instead of

H2S04 in superphosphate (SP) production

and using both with lower H)P04

concen-tration. to bioactivated Morrocan RP as raw materials increased the solubility of P in 2%

citric acid. It seemed that LCS could replace

H2S04 in the production of SP, and believed

to yield a more eco-friendly P fertilizer than conventional SP.

The objectives of this study were: (i) to determine phosphate solubilizing (PS)

acti-vity by A. niger BCC Fl94 on four types of

inorganic

P

compounds, (ii) to ･ｶｾｬｵ｡ｴ･＠ effect

of organic acids and phosphatase enzyme on RPs dissolution, and (iii) to conduct

agro-nomic evaluation of bioactivated CRP und

MRP for direct application in greenhouse experiment with cocoa, rubber, and oil palm seedlings as test crops.

Materials and Methods

Isolate

Aspergillus niger was isolated from Alfisols soils of Jeneponto, South Sulawesi,

Indonda (Goenadi et al., 1995). This soil

was highly weathered developing over vol-canic materials. Detailed characteristics of

the soil are, 31% sand, 27% silts, and 42%

clays; pH: 6.6 (H20), 5.3 (KC!); organic matter: 1.1% (C ), 0.1% (total N); 105 ppm P, 35 me/l OOg K; and clay mineral of smectite and kaoiinite. The' isolate was lod-ged in BaIitvet Culture Collection no. BCC F

194. Isolate was grown on Pikovskaya

medium in Petri dishes as a sou!'ce of ino-culum.

Phusphate solubilizing (PS) activity

Phosphate soluhilizing activity was assayed in 50 mL aliquots of standard Pikov-skaya's broth and modified PlkovPikov-skaya's

broth prepared by ｲ･ｾｬ｡｣ｩｮｧ＠ CalP04):

(19.9% P) with inorganic phosphate i.e.

AIP04 (50.8% P) and different rock

phos-phates, i.e. Cileung:;i (8.9% P) and Madura

(6.9% P). Cileungsi rock phosphate (CRP) and Madura ·rock phosphate (MRP) were ground (200 mesh) and oven dried. For this

purpose,

P

sourCes were sterilized separately

and then mixed with the sterile Pikovskaya medium.

Two pieces of inocula (0 3 mm) wcre

inoculateri into Pikovskaya liquid medium containing 0.125 % (w/v) resp:::ctive phos-phorus sources, incubated on a mechan i::al shaker at 100 rpm, 28°C for nine days. The dissolved P was then determined by applying

the molybdenum-b!ue method of Olsen &

Sommers (1982). Absorbance was measured

using

a

Spectronic 21 spectrophotometer at

693

urn.

Solubilization ofRPs in Pikovskaya medium

Modified Pikovskaya liquid medium supplemented with CRP or MRP of various concentrations (0,0.125,0.25,0.50, or 1.0 % (w!v)) was used. Inoculation was performed

by trans[.::rring two pieces of irocuia

(0 R mm) on to 100 mL ｅｲｬｾｭｮ･ｹ･ｲ＠ flask

containing 50 mL culture medium. The cul-tures were ir:cubated on a mechanical shaker

at 100 rpm (28cC) for nine days.

The growth of the fungus was measured in standard procedure by drying the decanted

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Santi et al.

veight at the end of incubation. Phosphorus

;olubilizing ability was detennined by using

he molybdenum-blue method of Olsen

&

ｾｯｭｭ･ｲｳ＠ (1982).

pH of the filtrates was

mea-;ured with Methrom pH meter and organic

.cid concentrations in the culture w<:re

､･ｴ･ｲｾ＠

nined by using HPLC

(0 0 I

N

H2

S0

4

mobile

,hase, 210 nm UV detector, 0.5 mLimin

lowrate,

at

50°C)

(Cunningham

&

Kuiark,

(992).

SfJect oforganic acids on RPs dissolution

This experiment was carried out to

;Iariry the relative strength of different types

0f organic acids in solubilizing P from the

eRP and MRP by using a method of IIImer

et .11. (1995).

Citrate, oxalate and gluconate

were added separately in different

concen-trations

(0, 0.05, 0.50, 1.0, 3.0

and

6.0

mM)

to 50

mL Pikovskaya mt!dium containing

0.125%

(w/v)

CRP or MRP, then incubated

for seven days on a mechanical shaker, at

100

rpm

(28°C).

P-concentration in

super-natant solutions was detennined Ilsing the

method of Olsen

&

Sommers

(I982).

Effect of phosphatase enzyme on RPs and inorganic P dissolution

The isolate of

A. niger

BCC F

194

was

grown in

50

mL modified Pikovskaya liquid

medium with vcrious levels of CRP or MRP

(0, 0.125, 0.25, 0.50

and

1.0%

(w/v)). The P

sources were sterilized separately and then

mixed with the sterile Pikovskaya medium.

The cultures were incubated for nine days on

a mechanical shaker at

100

rpm,

(280

C) for

the production of extracellular phosphatase.

Correlation between activity of phosphatase

(S0uciet

et al., 1980)

and P-solubilization

was detennined. One enzyme unit is the

amount which catalyses the hydrolysis of

1

!lmol of pNPP per min. under the

experi-mental condition.

Inactivation of phophatase in

I

iquid

cul-ture supernatant (LCS) of

A. niger

BCC

F

194

was studied at temperature of

40, 50, 60, 70.

80,

and

90

°

C.

Further,

50

mL LCS

or

A. niger

BeC

F J 49

contained inactivated

phosphatase was added with sterile

0.125%

(w/v) AIP04

and incubated on mechanical

shaker at

100

rpm, 28

°

C

for 24 h. Activity of

phosphatase enzyme (Souciet

el al., 1980),

P-solubilization, and concentration of organic

acid (mainly citric acid) were detennined.

Agronomic evaluation

of

bioaetivated CRP andMRP

Bioactivation was conducted by

reac-ting

8.5

mL LCS with the highest contents of

organic acids (mainly citric acid from

A. niger

BCe F194 isolate) and

28

mL

H3P04 52%

(v/v) on

55 g

RPs

(200

mesh)

(Goenadi

et al., 2000).

The most efficient

bioactivation of CRP and

MRP were

evaluated on the oasis of soluble P contents

in water and citric acid, as we II

as

perchlorate-extractable P content (SII

0029-73, 1984). Effectiveness of b0th granular and

powder biosuperphosphate prototypes in

substituting conventional P fertilizer,

i.e.

SP-36,

was determined on the basis of relative

agronomic effectiveness (RAE) (Mackay

el

aI.,

1984) in a completely random design

experiment with four levels of P dosages,

i.

e.

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Solubilization of insoluble phosphaJes by Aspergillus niger ...

Results and Discussion

Phosphate solubilizing (PS) activity

A. niger BCC F 194 solubilized Ca3

(P0

4)2,

CRP, and MRP, but showed poor

solubilization of AIP04 in nine-day

incu-bation period. The amount ofP solubilized by

A. niger BCC F194 was directly related to the

decrease in pH of the medium, except AIP04

treatment. These phenomena suggested that PS activity depends on the types and

cons-tanta solubility product (Ksp) of insoluble

phosphate (Ksp for AIP04 ｾ＠ 10.

30

and Ca3 (P04)2 - 2.0 x 10.2°) - as supposed by many

investigators (Mc Lean, 1976; Narsian et al.

1993; IIImer et al., 1995; Nahas, 1996).

Solubilization ofRPs in Pikovskaya medium

Employing a modified Pikovskaya

me-dium, A. niger BCC Fl94 produced oxalic

acid (3.75 mM), citric acid (2.0 mM), and gluconic acid (0.9 mM) as the main. organic acids from MRP and CRP as P source of

Pikovskaya medium lit 0 1.0% (w/v) level.

Regression analysis indicates that P-solubilization was highly positively corre-lated with organic acid concentration ( rMRP=

0.92** and fCRP=0.S5**) and ｮ･ｧ｡ｴｩｶｾｬｹ＠

correlated with the pH of the medium (rMRP=-0.87* and rCRP=-0.99**) (Figure 1). Organic acid concentrations were also strongly nega-tively correlated with the pH of the medium (rMRP =-0.99** and rCRP=-0.87*).

Effect oforganic acids on RPs dissolution

Production of organic acids is an impor-tant mechanis'11 for solubilizing inorganic ::>hosphate. Gluconic acid concentrations in Pikovskaya medium contained CRP and MRP as P source were comparatively con-;tant, whereqs the increased citrate and oxa-ate concentrations apparently reloxa-ated to the

increase in P-CRP and P-MRP concentrations

in solution (Figure 2). It indicates that citrate

and oxalate have strong influences to the solubilization of P-RPs, rCRP=O.96**, and rMRP=0.99** (citric acid) and rCRP=0.93** and

rMRP= 0.86* (oxalic acid). Oil the other hand,

no correlation between gluconk ;jcid wncen-tration and dissolution of CRP and MRP

(rcR.,,=0.6ns and rMRP= 0.1 ns) were found.

These phenomena lead to the assumption that

citric acid and oxalic acid produced by

A. niger BCC FI94 isolate are responsible [or lowering the pH of medium providing

pro-tons (W) to increase the P-RP solubilization.

In this case, there was relationship between the pKa values of the acids and the amounts of P released. Citric acid has a higher dis-sociation constant (pKa=3.14) than oxalic

acid (pKa = 1.25). Pohlman & McColl (198ii)

reported several factors that are important in determining the degree or rate of dissolution

of RPs,' i.e. : (i) rate of diffusion of organic

acid from bulk solution and diffusion of pro-ducts from the site of reactivity, (ii) contact time between the organic acids and mineral surface, (iii) degree of dissociation of organic acids, (iv) type and position of functional groups, and (v) chemical affinities of chelat-jng agents for the metals.

Table I. Solubilization of Ca3(PO.h, AIPO., Cileungsi rock phosphate (CRP), and Madura rock phosphate (MRP) by Aspergillus niger BCC FI94 in nine-day incubation

PS ability

P-Sources _(%) Initial pH Final pH

Cal(PO')l 65.0 7.3 4.3

AIPO. 7.8 6.0 2.8

Cileungsi rock 95.0 6.8 4.2 phosphate

[image:6.599.369.565.551.655.2]

(7)

Santi etal.

Cileungsi mck phosphate Madllra rock phosphate

7 6

7 ｲＭＭＭＭＭＭＭＭＭＭＭＭＭＭＭＭＭＭＭＭＭｾ＠

6

5 5

4 _:r: -1

:r: _c.

3

0.

3

2 2

I

oLI____________________

ｾ＠ 0

0 0.5 1.5 2 2.5 3 3.5 4 (j 0.25 0.5 0.75 1.25 J.S 1.75 2

ｏｲｾ｡ｬｬｩ｣＠ Acid (mM) Organic Acid (mM)

50 40

.2

30

10

O

:.c

_m

..g 30

ell Ｎｾ＠ｾ＠ . - CI) 20

:E .;:;

ｾＮＬＮ［＠

0 ,

-ｾ＠ 0 10

ｾ＠

o

ｾ

____

ＭＦｾｾ ____________ｾ＠

c

o

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

Oxalic Acid (111M)

40r,

.f?

30 '"

'"

:IJ

c::

o

Ｚｾ＠

:g

'0 _V">

0.872*

ｾ＠

c

ｏｾ ______________ｾｾｾｾ＠

o

2 3 4 5 6 7

pH

40

....

Ｎｾ

'"

"

ｾ＠

'0

() _0.5 1.5 2 2.5 3 3.5 4

Oxalic Acid (mMl

50

I

40

30

"

'"

Ｇｾ＠

20

"-'"""

C

ｲ］ＭｏＮＹＹＹＢｾ

10

O

0 2 4 5 7

[image:7.599.168.563.119.549.2]

pH

Figure I. The correlation between pH and organic acid production [top). P solubilizing ability of Aspergilus niger BCC

FI94 lind oxalic acid production (middle), and P SOlubilizing ab;lity and pH (bOUOln) with rock phosphate from

Madura and from Cilcul1gs: as

r

sources in Pikovskaya medillm

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Solubilization of insoluble phosphates by Aspergillus niger .. _

Madura rock phosph;;te

70

1

Ys 9,9409x -;- 1.5739

rs = 0.9992"

60

1

,

/ /

/

Yo 0.1904x +4,7402

ro ｏｊｾＶＱＷＧ＠

Y ('

=

0.0672x + 4_25:' rG = 0_1049"

:/) 20 _{• Citric Acid}

.6. Oxalic Acid

10

II •

Gluconic Acid

lP=::::::::i:====!

O'F,r.,.-o

2 4 6

Organic Acid Concentration (mM)

140

120

ｾ＠

c

.e

E

iuG

ｾ＠

ｾ＠ 8(; ::::

.,

u

c

0

u

A. ₆₀

;,

:3

::l

0

Vi

20

0

Cileungsi rock phosphate

Ys = 17,964x + 9.5586

rs = 0,'.1600"

Yo =8.2758x1.3416 /

ro = 0,9318*'

YG = 0

ｉｾ＠

73x + 1;,8696/

rc 0.6681 /

,;

1

40 I

•

• Citric Acid

lJ. Oxalic Acid

6. • Gluconic Acid

2

ｾ＠

so

c.

e

ｾ＠

40

;:;

o

ｾ＠

:,) 30

:c

'"

:::J

"0

0 4 6

Organic Acid Concentration (mM)

'igure 2. Relationship between concentration of selected org2nic acids (citric, oxalic, or gluconic) and

solubilization of rock phosphate from Madura and Cileungsi in modified Pikovskaya medium

ｾｬｦ･｣ｴ＠ ofphosphatase enzyme on RPs and

'1organic P dissolution

Phosphate solubilization mediated by lhosphatase enzyme is believed to be taken Jlace for organic P sources (Bishop et al.,

994). There was no correlation between lctivity of phosphatase enzyme and P

;olubilization (rcRP=0.39"S and rMRP=O.22"'),

,s well as with P concentration (rMRP=O.39n$ md rCRP=0.23"S). The phosphatase activity 'ikovsakaya medium decreased at 80°C

15 U/mL) and 90°C (5.8 U1mL) (Figure 3A).

'::omplete denaturation of protein by heating Jrocess possibly occurred at 80°C, therefore lhosphatase enzyme was inactivated. The

corr"lation between phosphatase activity and AI P04 solubilization indicates that there was

no significant Psolubilizing value In liquid culture supernatant of A. niger BCC F 194 unheated (UH) and heated (H) at 80° C, although the phosphatase activity was dif-ferent, i.e. 127.3 U/mL (UH) and 7.9 U/mL (H), respectively. (Figure 3B). On the other

hand, the citric acid con<.entration was

relatively stahle at 020 minute of hearing periods (Figure 3C). These results were in agreement with the assumpHon that phos-phate solubilization mediated by phosphatase enzyme is taken place for organic P sources (Traina el oj., 1986; Bishop et 01., 1994; Sigh

(9)

Santi et a!.

200 _A

a

150

Ｚｾ＠

u

ｾ

.,

'"

",....l

100

ｾ＠ E

.::;;:-

"'-ｾｾ＠

<.l 50

c::

0

100

Temperature (He)

• 300 _B

250 ..",

:::r 0

"t:J

'"

200 :::r

ｾＧＢ＠

1= g 150 ｓＺＺｾ＠

ｾ＾Ｍ

g

100 _ｾＺ＠

Q

50

0

0 5 10 15 20 25

Heating Period (l11inute) at BOile

0

y -3.53x + 317.4 Rl_ 0.881.7

20 40 60 80

30

'"

0 25

·E

₂₀

;::

''

t )

RE

15

P- o. 0.

0 " 10

:E

::J

<5 ₅

C/l

0

+ Soluble P

0 Ph0sphatase Activity

t:

c

.g

O.B

f

t:

g

•

..

0

t )

0.6

c

t: _.::J

0 _E

"t:J

-. on

gg

0.4 'ct )

'"

on 0.2

0

0 10 15 20 25

[image:9.601.210.529.78.612.2]

ｈｾ｡ｴｩｮｧ＠ Period (,ninute) at 8U"e

Figure 3. Effect of temperature (A). and heating period (minute) ｾｴ＠

so·e

011 acti·.'ity or phosphatase enzyme (8). and effect of heating period M 80· C on organic acid (citric acid) concentration (e)

(10)

•

Solubilization 0/ insoluble phosphates by Aspergillus niger, ,

Table 2. Relative agronomic effectiveness of constructed effect ofbiosupcrphosphate (SPab) of cocoa, rubber, and oil

palm seedlings dry weight '

Relative Agronomic Effectiveness (%) Types of

biosllper- Form Rate

cocoa rubber oil palm

phosphate

seediings seedlings seedlings

CHeung:>i granule 50 62.5 b') 65.8 c 120.2 abc

Cileungsi granule :00 96.1 ab 96,9 bc IOLJ abc

ｃｩｬ･ｵｮｾｳｩ＠ granule 150 89.2 ab 166.9 a 77.9 c

CiJeungsi powder 50 SO.5 ab 146,9 ab 1142 abc

Cileungsi pOWder 100 80.1 a b 137,5 db 93J abc

Cileungs! powder i50 59,7 b 121).9 abc 92.5 abc

Madura granule 50 69.5 b 104.2 abc 166.9 a

Mad<Jra granule 100 85.5 ab 103.9 abc 127.9 abc

Madura granule 150 115.2 a 150.9 ab 153.9ab

Madura powder 50 93.1 ab 61.1 c 83.7 be

Madura powder 100 113.8 a 97,6 be 97,9 abc

Madura powder 150 78,6 ab 99.7 be 1103 abc

Standard (SP-36) 100 100.Oab 100,0 be 100,0 abc

Note: *) Figures in each column/ollowed by the same leller (s) are not Significantly different (P<O.05) according to Duncan's multiple range lest

Agronomic evaluation of bioactivated CRP and MRP for direct application in green-house experimem

Application of biuactivated CRP and MRP resulted in significant increase of leaf

number dod height of the three-month old

co-coa and oil pCllm seedlings. For rubber seed-lings, significant responses have been obser-ved staning at four months after treatment (P<0.05).

There were no significant different-ces between conventional SP and ｢ｩｯｳｾｰ･ｲﾭ

phosphate (SPab) applied on height, leaf number, girth, and dry weight of cocoa, rubber, and oil palm seedlings (unpublished

data). At the end C'f the experiment (four

months after treatment for cocoa and rub!Jer, and six months for oil palm seedling), CRP

and MRPoriginating SPab In granular and

powder form applied at 100% rate equivalent to standard SP application indicated a higher value of relative agronomic effectiveness

(RAE) (Table 2).

Conclusions

A. niger DCC F 194 performed high abilities in solubilizing inorganic phosphate

(>50%), i.e. Ca3 (P04)2 , CRP, and MRP, but

not for AIP04• Citric and oxalic acids were

important components responsible for

phos-phate solubilization by A. niger BCC FJ 94.

Oxalic acid was the main organic acid

pro-duced by A. niger BCC F194 in modified

Pikovskaya medium', for nine days incubation at 100 rpm, 2S°C. Citrate and oxalate had

strong influences to the solubilization of CRP

and MRP. There was no correlation between phosphatase activity and solubilization of inorganic phosphate. Results of cocoa, rub-ber, and oil palm seedlings experiments in greenhouse showed that the prototype pro-ducts from bioactivation (biosuperphosphate) showed a comparable relative agronomic effecttiveness value to that of the conven-tional SP (SP36).

[image:10.601.163.564.168.352.2]

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Santi ct al.

Acknowledgment rocks with a P soluLilizing fungus. Soil. Sci.

The authors wish to thank the Riset Unggulan Terpadu VII Project Management

of the Ministry..ofResearcll ｾｮ､＠ Technolo.gy

for financial support (Contract No.: 17/SPK!

RUT/BPPT/IV/2000).

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•

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Menara Perkebunan, 2000 68(2)

Solubilization of insoluble phospates by Aspergillus niger

JURNAL PENELITIAN BIOTEKNOLOGI PERKEBUNAN

Solubilization of insoluble phosphates by

veight at the end of incubation. Phosphorus

ｾｯｭｭ･ｲｳ＠ (1982).

pH of the filtrates was

;Iariry the relative strength of different types

P-concentration in

(w/v)). The P

(S0uciet

Further,

reac-ting

in water and citric acid, as we II

7 ｲＭＭＭＭＭＭＭＭＭＭＭＭＭＭＭＭＭＭＭＭＭｾ＠

ｲ］ＭｏＮＹＹＹＢｾ

RUT/BPPT/IV/2000).

CONTENTS