Jurnal Penelitian Bioteknologi Perkebunan

ISSN 0215 - 9318

JURNAL PENELITIAN BIOTEKNOLOGI PERKEBUNAN INDONESIAN JOURNAL OF BIOTECHNOLOGY RESEARCH ON ESTATE CROPS

Menara Perkebunan 2000 68(2) 37 47

Solubilization of insoluble phosphates by Aspergillus niger

Pelarutanfosfat sukar larUE oleh Aspergillus niger

LAKSMITA P SANTI I) D H GOENADr I) SISWANTO i

)

I SAILAI-e) amp ISR0I3)

I) Biotechnelogy Research Unit for Estate Crops Bogor 1MSI Indonesia 2) Bogor Agricultural University Bogor Indonpsia

3) Graduate School Boger Agricultural University Bogor Indonesia

Ringkasan

Penggunaan angsung fosfat aam (FA) ke daam tanah sebagai sumber pupuk P teah dilashykukan seama bertahun-tahun melalui beberapa macam cara penggunaan Kualitas FA di Indoshynesia umumnya rendah dan ketersediaan bahan baku yang berkualitas untuk produksi pupllk fosfat terarut 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 oell Aspergillus niger BCC FI94 belum banyak diteiti Satu seri penelitial laboralorium lelah dilaksanashykan 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(POJ2 dan AIP04 Kelarutan P anorganik lersebut bershyhubungan 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 oksaat (375 mM) asam silral (20 mM) dan asam glukonat (OQ mM) Kelarulan FA CilelIngsi Ibih besar dibandingkan dengan FA Madura dan kelarulan CalPO)2 lebih besar dibandingkan kelarlltan AIP04bull Tidak ada korelasi anlara kdarutan P anorganik dengan aktivitas enzim fosfatase walaupun aktivitas enzim fosfashylase 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 protoshylipe pupuk biosuperfosfa dengan bahan baku FA Cileungsi dan Madura bentllk granul malplln serbllk memiliki niai keefektifan agronomi yang relatif menyamai SP-konvensional

Summary

The direct application of rock phosphate (RP) to soils as a source of phosphorus (P) fertishylizer has been employed with varying popular methods over the ycars The RP in Indonesia has jow quality for plant fertilization and the Hyaishylability of the raw material with good quality for production of soluble phosphate fertilizers is limited iany common soil microbes that can dissolve insoluble inorganic phosphate have been extensively studied However there is lillie inforshymation on mechanism or P-solubilization from local RP of Indonesia and inorganic P by Aspershygiflus niger BCC F194 isolated from tropical aeid soils A laboratory study was fGnducted to determine the ability of A niger Bee F 194 to solubilize P Agronomic evaluation ofbioactivaLcd local RP ie Cileungsi and Madura phosphate rocks (CRP and MRP) for direct application as also conducted in greenhouse experimcn 1 lIiger BeC FI94 was able in solubilizing dilTerent types of hardly-soluble phosphates ie CRP and rvlRP Ca3(P04)2 and AIP04 compounds Inorganic P solubilization was directly correlalC(i to the increasing proton (H+) activities causing plJ to

37

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 (375 mM) citric acid (20 mM) and gluconic acid (09 mM) The solubiization of Cileungsi RP was higher than that of Madura RP and the solubilization of C3(P04h flS better than that of AIP04 There is no correlation between solubilization of inorganic P and en~yme activities although high level of activity of phosphatase enzyme Vas detectaJe in the medium A biosuperphosphate formula had heen constructed by reacting 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 producshytion The composition of these rock phosshyphates 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 laquo25 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 enshyhancing the rock phosphate availability is to increase its solubility by treating with inorshyganic acids mainly sulphuric acid and phosshyphoric acid but this approach is not applishycable 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 solushybiliLe insoluble forms of inorganic phosphatic compounds [n vitrc studies with microbial isolates from ~oil indicated that fungi were more efficient in thc solubilization of inorshyganic 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 particularshyly A niger and some Penicillium sp which have been tested i1 fermentation systems or inoculated directly into soil in order to solushybilize rock phosphate (Vassilev et al 997 V asi leva et al 1998)

Acidification of soil is a consequence of natural and anthropogenic processes (Ulrich amp 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 fixashytion of plant-available P on the other hand Hardly-soluble aluminum phosphates are forshymed and become the largest P-fraction in many acidic soils (McLean 1976) AIP04bull i1 the contrary to calsiui7l phosphate will never play an important role il1 ameliorating soils Leaving that out of consideration the imporshytance of Alr04 solubilization fOf soil formashytion mineral transformation and AI-toxicity is obvious (lllmer et al 1995) In this invesshytigation pure insoluble inorganic phosrhates i e aluminum phosphate (A IP04 ) and ca 1shycium phosphate (CaJ(P04)2) have been used for expressing phosphate olubiizillg activity of A 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

38

Soiubilizatiol if insoluble phosphates by Aspergillus niger

a growing interest in ways of manipulating such rock to obtaIn a more valuable product ie partially acidulating RPs (Goenadi 1990 Rajan amp Ghani 1997) reacting with synshythetic organic acids (Sagoe et al 1998) or natural organic acids (Singh amp Amberger 1998) and decreaing 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 concenshytration 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) actishyvity by A niger BCC Fl94 on four types of inorganic P compounds (ii) to ev~luate effect of organic acids and phosphatase enzyme on RPs dissolution and (iii) to conduct agroshynomic 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 volshycanic materials Detailed characteristics of the soil are 31 sand 27 silts and 42 clays pH 66 (H20) 53 (KC) organic matter 11 (C ) 01 (total N) 105 ppm P 35 mel OOg K and clay mineral of smectite and kaoiinite The isolate was lodshyged in BaIitvet Culture Collection no BCC F 194 Isolate was grown on Pikovskaya

medium in Petri dishes as a souce of inoshyculum

Phusphate solubilizing (PS) activity

Phosphate soluhilizing activity was assayed in 50 mL aliquots of standard Pikovshyskayas broth and modified Plkovskayas broth prepared by re~lacing CalP04)

(199 P) with inorganic phosphate ie AIP04 (508 P) and different rock phosshyphates ie Cileungi (89 P) and Madura (69 P) Cileungsi rock phosphate (CRP) and Madura middotrock 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 0125 (wv) respctive phosshyphorus sources incubated on a mechan ial shaker at 100 rpm 28degC for nine days The dissolved P was then determined by applying the molybdenum-bue method of Olsen amp 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 (00125025050 or 10 (wv)) was used Inoculation was performed by trans[rring two pieces of irocuia (0 R mm) on to 100 mL Erl~mneyer flask containing 50 mL culture medium The culshytures were ircubated 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 mycelium in an oven at 70 0 C to constant

39

Santi et al

veight at the end of incubation Phosphorus olubilizing ability was detennined by using he molybdenum-blue method of Olsen amp ~ommers (1982) pH of the filtrates was meashyured with Methrom pH meter and organic cid concentrations in the culture wltre deter~ nined by using HPLC (0 0 I N H2S04 mobile hase 210 nm UV detector 05 mLimin lowrate at 50degC) (Cunningham amp 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 concenshytrations (0 005 050 10 30 and 60 mM) to 50 mL Pikovskaya mtdium containing 0125 (wv) CRP or MRP then incubated for seven days on a mechanical shaker at 100 rpm (28degC) P-concentration in supershynatant solutions was detennined Ilsing the method of Olsen amp 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 0125 025 050 and 10 (wv)) 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 experishymental condition

Inactivation of phophatase in I iquid culshyture supernatant (LCS) of A niger BCC F 194 was studied at temperature of 40 50 60 70 80 and 90 deg C Further 50 mL LCS or A niger BeC F J 49 contained inactivated phosphatase was added with sterile 0125 (wv) AIP04 and incubated on mechanical shaker at 100 rpm 28 deg 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 reacshyting 85 mL LCS with the highest contents of organic acids (mainly citric acid from A niger BCe F194 isolate) and 28 mL H3P04 52 (vv) 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 0029shy73 1984) Effectiveness of b0th granular and powder biosuperphosphate prototypes in substituting conventional P fertilizer ie SPshy36 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 0 50 100 and 150 to that of recommended SF-36 dosage The RAE () was evaluated on the basis of seedlings dry weight that c(llcuiated from a greenhouse experiment using cocoa rubber and oil palm seedlings grown on Cikopomayak Uitisols West Java (pH 47 181 ppm P20 S (Olsen test) total N 010 organic matter 29 (C) and 01 (N))

40

Solubilization of insoluble phosphaJes by Aspergillus niger

Results and Discussion

Phosphate solubilizing (PS) activity

A niger BCC F 194 solubilized Ca3 (P04)2 CRP and MRP but showed poor solubilization of AIP04 in nine-day incushybation 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 consshytanta solubility product (Ksp) of insoluble phosphate (Ksp for AIP04 ~ 1030 and Ca3 (P04)2 - 20 x 102deg) - 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 meshydium A niger BCC Fl94 produced oxalic acid (375 mM) citric acid (20 mM) and gluconic acid (09 mM) as the main organic acids from MRP and CRP as P source of Pikovskaya medium lit 0 10 (wv) level

Regression analysis indicates that Pshysolubilization was highly positively correshylated with organic acid concentration ( rMRP= 092 and fCRP=0S5) and negativ~ly correlated with the pH of the medium (rMRP=shy087 and rCRP=-099) (Figure 1) Organic acid concentrations were also strongly negashytively correlated with the pH of the medium (rMRP =-099 and rCRP=-087)

Effect oforganic acids on RPs dissolution

Production of organic acids is an imporshytant mechanis11 for solubilizing inorganic gthosphate Gluconic acid concentrations in Pikovskaya medium contained CRP and MRP as P source were comparatively conshytant whereqs the increased citrate and oxashyate concentrations apparently related 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=O96 and rMRP=099 (citric acid) and rCRP=093 and rMRP= 086 (oxalic acid) Oil the other hand no correlation between gluconk jcid wncenshytration and dissolution of CRP and MRP (rcR=06ns and rMRP= 01 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 proshytons (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 disshysociation constant (pKa=314) than oxalic acid (pKa = 125) Pohlman amp McColl (198ii) reported several factors that are important in determining the degree or rate of dissolution of RPs ie (i) rate of diffusion of organic acid from bulk solution and diffusion of proshyducts 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 chelatshyjng agents for the metals

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

PS abilityP-Sources Initial pH Final pH()

Cal(PO)l 650 73 43 AIPO 78 60 28 Cileungsi rock 950 68 42 phosphate Madura rock 576 76 63 phosphate

41

Santi etal

Cileungsi mck phosphateMadllra rock phosphate

7

6 7 r---------------------~

6

5 5

4 -1rr c 30 3

2 2

IoLI____________________~ 0

0 05 15 2 25 3 35 4(j 025 05 075 125 JS 175 2 Or~allic Acid (mM) Organic Acid (mM)

5040 2

30

10

O

c mg 30

ell

~ ~

- CI) 20E ~ 0shy

~ 0 10 ~

o ~____-amp~~____________~ c-

o 01 02 03 04 05 06 07 08

Oxalic Acid (111M)

40r------------------------ f

30 - IJ co ~ g 0

Vgt-0872 ~ c-

O~______________~~~~

o 2 3 4 5 6 7

pH

40

~ ~ 0

() 05 15 2 25 3 35 4

Oxalic Acid (mMl

50 I 40

30 ~ 20 shy-

C r=-O999~10

O 0 2 4 5 7

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 ablity and pH (bOUOln) with rock phosphate from Madura and from Cilcul1gs as r sources in Pikovskaya medillm

42

Solubilization of insoluble phosphates by Aspergillus niger _

Madura rock phosphte

70 1 Ys 99409x -- 15739

rs = 09992 60 1

Yo 01904x +47402

ro OJ~617

Y( =00672x + 4_25 rG = 0_1049

) 20 bull Citric Acid

6 Oxalic Acid

10 II bull Gluconic Acid

lP=i==== OF-----r---------- shy

o 2 4 6

Organic Acid Concentration (mM)

140

120

~

c e E iuG

~ ~ 8( u c 0 u A 60 3 l

-0 Vi

20

0

Cileungsi rock phosphate

Ys = 17964x + 95586 rs = 01600

Yo =82758x-13416 ro = 09318

Y G = 0 I~ 73x + 18696 rc- 06681

140 I bull

bull Citric Acid lJ Oxalic Acid

6 bull Gluconic Acid

2

~ so c e ~ 40

o ~ )

30

cJ 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

~lfect 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=039S and rMRP=O22) s well as with P concentration (rMRP=O39n$ md rCRP=023S) The phosphatase activity ikovsakaya medium decreased at 80degC 15 UmL) and 90degC (58 U1mL) (Figure 3A) omplete denaturation of protein by heating Jrocess possibly occurred at 80degC therefore lhosphatase enzyme was inactivated The

corrlation between phosphatase activity and AI P04 solubilization indicates that there was no significant P-solubilizing value In liquid culture supernatant of A niger BCC F 194 unheated (UH) and heated (H) at 80deg C although the phosphatase activity was difshyferent ie 1273 UmL (UH) and 79 UmL (H) respectively (Figure 3B) On the other hand the citric acid conltentration was relatively stahle at 0-20 minute of hearing periods (Figure 3C) These results were in agreement with the assumpHon that phosshyphate solubilization mediated by phosphatase enzyme is taken place for organic P sources (Traina el oj 1986 Bishop et 01 1994 Sigh amp Amberger 1998)

43

Santi et a

200 A a

150~ u ~

l 100 ~ E shy shy~~ ltl 50c

0 100

Temperature (He)

bull 300 B 250

r 0

tJ 200 r ~ 1= g

150 S~ ~gt-

g100 ~

Q

50

0 0 5 10 15 20 25

Heating Period (l11inute) at BOile

0

y -353x + 3174 Rl_ 08817

20 40 60 80

30

0 25

middotE 20

- t)

RE 15 Pshy o

0 0 10

E J

lt5 5Cl

0

-+- Soluble P

-0- Ph0sphatase Activity

t cg OB ft

g bull 0 t)

06c t J 0 E

tJ shy- ongg 04

ct)

on 02 0

0 0 10 15 20 25

H~ating Period (ninute) at 8Ue

Figure 3 Effect of temperature (A) and heating period (minute) ~t somiddote 011 actimiddotity or phosphatase enzyme (8) and effect of heating period M 80middot C on organic acid (citric acid) concentration (e)

44

bull

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

CHeunggti granule 50 625 b) 658 c 1202 abc Cileungsi granule 00 961 ab 969 bc IOLJ abc Cileun~si granule 150 892 ab 1669 a 779 c CiJeungsi powder 50 SO5 ab 1469 ab 1142 abc Cileungsi pOWder 100 801 a b 1375 db 93J abc Cileungs powder i50 597 b 121)9 abc 925 abc Madura granule 50 695 b 1042 abc 1669 a MadltJra granule 100 855 ab 1039 abc 1279 abc Madura granule 150 1152 a 1509 ab 1539ab Madura powder 50 931 ab 611 c 837 be Madura powder 100 1138 a 976 be 979 abc Madura powder 150 786 ab 997 be 1103 abc Standard (SP-36) 100 100Oab 1000 be 1000 abc

Note ) Figures in each columnollowed by the same leller (s) are not Significantly different (PltO05) according to Duncans multiple range lest

Agronomic evaluation of bioactivated CRP and MRP for direct application in greenshyhouse experimem

Application of biuactivated CRP and MRP resulted in significant increase of leaf number dod height of the three-month old coshycoa and oil pCllm seedlings For rubber seedshylings significant responses have been obsershyved staning at four months after treatment (Plt005) There were no significant differentshyces between conventional SP and bios~pershyphosphate (SPab) applied on height leaf number girth and dry weight of cocoa rubber and oil palm seedlings (unpublished data) At the end Cf the experiment (four months after treatment for cocoa and rubJer and six months for oil palm seedling) CRP and MRP-originating 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 (gt50) ie Ca3 (P04)2 CRP and MRP but not for AIP04bull Citric and oxalic acids were important components responsible for phosshyphate solubilization by A niger BCC FJ 94 Oxalic acid was the main organic acid proshyduced by A niger BCC F194 in modified Pikovskaya medium for nine days incubation at 100 rpm 2SdegC 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 rubshyber and oil palm seedlings experiments in greenhouse showed that the prototype proshyducts from bioactivation (biosuperphosphate) showed a comparable relative agronomic effecttiveness value to that of the convenshytional SP (SP-36)

shy

45

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 MinistryofResearcll ~nd Technology for financial support (Contract No 17SPK RUTBPPTIV2000)

References

Babare M PWG Sale N Fleming DL Garden amp D Johnson (1997) The agroshynomic effectiveness of reactive phosphate rocks 5 The effect of particle size of a moderately reactive phosphate rock Aus 1 Exp Agric 37969middot984

Bishop ML AC Chang amp RWK Lee (1994) Enzymatic mineralization of organic phosshyphorus in a volcanic soil in Chile Soil BioI Biochem 157238-243

Cunningham JE amp C Kuiack (1992) Producshytions of citric and oxalic acids and solubilishyzations of calcium phosphate by Penicillium bilaji App Env Microbial 58(5) 1452shy1458

Gerke L (1992) Phosphate aluminium and iron in solution of three different ~ois in relation to varying (uncentration of citric acid 1 Plant Nutr Soil Sc 155 339-343

Goenadi DI-I (1990) Effect of acidulation on the mineralogical characteristics of a comercial phosphate rock lndon 1 Trop Agric 2 1shy5

Goenadi DH R Saraswati NN Nganro amp lAS Adiningsih I 995) Mikroba pelamt hara dan pemantap agregat dari beberapa lanah tropika basah Menaru Perkebunan 63(2)60-66

Goenadi DH RA Pasaribu Isroi H Hartono amp R Misman (1999) Phosphate-solubilshyizing fungi isolated from tropical forest soils Menara Perkebunan 67( 1)40-51

Goenadi DI-I Siswimto amp Y Sugiarto (2000) Bioactivation of poorly soluble phosphate

Soc Am J 64 927-932

Hammond L L SH Chien amp AU Mokwunye (1989) Agronomic value of unacidulated and partially acidulated phosphate rocks indigenous to the tropics Adv Agr 40 89shy140

JIlmer P A Barbato amp F Schinncr (1995) Solubilization of hardly-soluble AIPOd wth P-solubilizing micloorganisms Soil Bhl Biochem 27 (3) 265-270

Mackay ADIK Syers amp PEH Gregg (1984) Ability of chemical extraction procedures to assess the agronomic effectiveness of phosshyphate rock materials New Zealand f Agric Res 27 219-230

McLean EO (I 976 Chemistry of soil alumishynium Communications in Soil Sci amp Plant Anal 7 619-636

Moersidi (1999) Fofat Alam sebagai Bahan Baku dan Fupllk Fosfat Bogor Pusat Penelitian Tanah dan Agroklimat pp 82

Narsian V 1 Thakkar amp HB Patel (1993) Solubilization of natural rock phosphates and pure i1soluble inorganic phosphates by Aspergillus awamori Ind J Exp BioI 31 747-749

Olsen SR amp LE Sommers (1982) Phosphorus In Page AL RH Miller amp DR Keeney (Eds) Methods of Soil Analysis Agronomv series No9 Madison American Society of Agronomy p 403-430

Pohlman AA amp IG McColl (1986) Kinetics of metal dissolution from forest soils by soluble organic acids J Environ Qua 1586-92

Rajan SSS amp A Ghani (1997) Differential influence of soil pH on the availability of partially sulphuric and phosphoric acidulated phosphate rocks 2 Chemical and scanning electron microscopic studies Nut Cyc Agroecosyst 48 (3)171-178

46

Solubilization of insoluble phosphates by Aspergilus nii~r

Sagoc Ct T Ando K Kouno amp T Nagaoka (1996) Response of italian ryegrass to phosphorus in organic - acid treated phosshyphate rocks 1 Fac Appl Bioi Sci 35 199shy209

Singh CPo amp A Affiberger (i 998) Organic acids and phosphorus solubilization in straw COIllshy

posted with rock ph0sphate Bioresollrce Technol 63( I) 13-16

Souciet G 1 Attias amp 1 d Auzac (1980) A neutral cytoplasmic phosphatase from the latex of Hevea brasiliensis Phytochem 19 20Q9-2102

Thomas GV MV Shantaram amp N Saraswathy (1985) Occurrence and activity of phosshyphate-solubilizing fungi from coconut planshytation soils Plant amp Soil 87 357middot364

Traina SJ G SJosito D Hesterberg amp u Kakafi (1986) Effects of pH and organic acids on orthophosphate solubility in acidic montmorillorltic soil Soil Sci Soc Am J 5045-51

Ulrich B amp ME Sumner (1991) Suil Acidity Berlin Springa-Verlag

Vussiiev N M Vasslleva amp R Azcon (1997) Solubilization of rock phosphate hy immoshybilized Aspergillus niger Bioresource iechnol 59 1-4

Vassileva M R Azcon J Barea amp N Vassilev (1998) Appication of an encapsulated filamentuus fungi in solubilization of inorganic phosphate 1 Biotechnoi 63 (I) 67middot72

47

bull

Menara Perkebunan 2000 68(2)

CONTENTS

Research Reports Page

Transfonnation of Cojlea arabica using chitinase gene and regeneration of plantlets from transformed-zygotic embryos (Transformasi Com~a arabic a menggunakarl gen kinitase dan regenerllsi planlet dad embrio zigotik haLltpound transfarmasi) shy A Budiani T Chaidamsari Priyono S Mawardi amp Siswanto I - 11

Overexpression of chitinase gene with a GC-rich -synthetic enhancer in tobacco plant (Nicotiata tabacum) (Overeskpresi gen kinitase dengan enhancer sintesis kaya GC pada tanaman tembakau (Nicotiana tabacum) D SantoslJ T Chaidamsari A Budiani H Minarsh S Dwi Utomo amp Siswanto 12 - 20

Development of tobacco plant cells in the presence of kanamycin at various levels for transgenezis (Perkembangan sel tanaman tembakau pado berbagai konsentrasi kanamisin untuk transgenesis) shy D Santoso Ferry I Cugito amp H Minarsih 21 28

Extraction and characterization of humic acid from plantations solid organic waste composts (Ekstraksi dan karakterisasi asam humat dari kompos limbah padat organic Perkebunan) - Laksmita P Santi DH Goenadi H Widiastuti N Mardiana amp Isroi 29 36

Solubilization of insoluble phosphates by Aspergillus niger (Pelarutan fosfat sukar larut oleh Aspergillus niger) - Laksmita P Santi DH Goenadi Siswanto I Sailah amp Isroi 37 47