Zbl. Mikrobi ol. 138 ( 1 98;~ ), 437-442

[University College of Agri culture, ~1icrobiolog~el\ l L" L" rl\l iJi'Y, Caleuttn Univrr:1it:y,-Gn)r:uHfi.India, and Department of Agricul tura l Chemist ry and Soil Science , Bidhan Chandra Krishi .Viswa Vid yalaya, Kalyani, I ndia]

Alluvial Soill\1icroorganisms Capable of Utilizing InsolubleAluminium Phosphate as a Sole Source of Phosphorus

S. B ANI K and B . K . DEY

Summary

F rom an Indian alluvial soil (Fluvaquent ), 5 bacteria - all of the genus Bacillus, 5 actino ··mycetes - all of the genus Stre ptomyces, and 6 fungi - 1 of the genus P enicillium, 3 of th egenus Chaetomium, and 2 unidentified ones - were isolated on sucrose -a luminium phospha teag ar plates. All of them , with th e exce p t ion of B aci llus sp . AA85, Streptomyces sp. AAS 5, and,Chaetomium sp. AAF5, solubilized Ca3(P04h wit h greater efficiency than AlP04• B acillus coagu·lans (B. 7659) AAB 2 showed highest efficiency in solubilizing Ca3(P 0 4)2' foll owed by Penicillium.sp. AAF6 • Ba cillu s sp. AABl' B . subti li« (B -7660), B . megatherium (B.766 1) AAB 4, St reptomycessp . AAS4, a nd Penicill ium sp . AAF6 were unable to solubilize AlP04 in detectable amount , ex­ceed ing their nu tritional demand, while S treptomyces sp , AAS:; beh aved simila rly with Ca3(P04)2"

Th e orga nic ac ids produced in t he culture b roth were oxalic, maleic, mali c a nd 2-ketogluconi c'acids. P roduction of 2-ketogluco n ic acid had the high est eff iciency in solubilizat ion of in solubleinorganic phos phates. Production of oxalic acid was found to be less efficient in AlP04 solubili­za ti on . Th e a mo unt of free organic ac ids, presen t in the growth medium during solubilization , was.not. always indicative of phosphate solubilizat ion .

Zusammenfassung

Aus einem indischen Allu vial-B oden (F luvaquent) wurden 5 Ba kt erien (B acillus-Arten),.5 Aktinomyzeten (St reptomyces·Arten) und 6Pilze (Peni cillium.Arten , :l Chaetomi um-Arte n und2 ni cht-ident.if'izierte) auf Sukrose.Aluminiumphosphat-Agar isoliert . Mit Ausnahme von B acillus.sp . - AAB 5 , St reptom yces sp. - AAS5 und Chaetomium sp . - AAF5 losten alle Mikroorganism enCa3(1'0 4 ) 2 besser als AlP 0 4 • Ba cillus couquluns (B-7659) - AAB 2 wa r bei der Losung von Ca3(1'04) 2,

am wirksamston, gefo1gt von P enicillium sp . - AAF6• Bacillus sp . - AABl' B. subtili s (B -7660) ­AAB3, B . megatherium (B- 7661) - AAB 4, Streptomyces sp. - AA84 und P enicillium sp. - AAF6.

waren ni eht fahig, A11'0 4 in einer Men ge zu losen, die uber ihren eigene n P -Be darf hinausging,.wah rend S trep tomyces sp . - AAS5 iihnlieh wie bei Ca3(1'04) Z re agierte . Die im Kulturmedium er ­zeug ten organischen Sauren wa ren Oxa lsauro, Maloinsaure, Apfelsiiu l'e und 2-Ketoglukonsaure..Letzter e war an der Losung von unl oslichen anorgan ischen Phosphaten a m st iirksten beteiligt.Ox alsaure war bei der L osung vo n Al1'04 weni ger wirksam. Der in Nahrmedium vo rhandene Ge­halt a n frei en organisehen Sauren wah rend des Losungsprozesses war nicht immer ein Indikat.or ­fiir die P.l\!obilisicl'llng.

Soil pho sphates in the tropics are predominantly inorganic and are present ininsolub le states, of which aluminium pho sphate is one of the major form s (RUSSEL.

19n ). An appreciable number of the diverse gene ra of soil popu lation possessingphosphate solubilizing abi lit y (HAYMAN 1975) may be though t to be act ive on AlP04(BANIK and D E Y 1982). Elaborati on of aliphat ic dicarboxylic, hydroxy and ketoacids by the responsible organisms, have been found t o be the maj or means of so­lubilizing insolub le inorganic phosphates (J OHN STON 1954, BANIK and D EY 1981)..

4:38 S. BEIR and B. K, Ptiy

The' present study has been an attempt for characterization of the microflora of anair-dried Indian alluvial soil (Fluvaquent, DOLUI 1977), active in solubilizing AIP04,

especially in terms of phosphate solubilizing potentiality and production of aliphaticorganic acids responsible during solubilization.

Materials and Methods

The soil sample was collected from Calcutta University Agricultural Farm, Baruipur, District24 Parganas, West Bengal, India. The soil was characterized as Fluvaquent (DoLUI 1977), accord­ing to Seventh Approximation. Air dried and 2 mm sieved soil samples were used for the presentstudy.

Enumeration of microorganisms

Enumeration of total bacteria, actinomyeetes, fungi, and aluminium phosphate solubilizingorganisms (including bacteria, aetinomycetes and fungi) were done according to the method adopt­ed by BANIK and DEY (1981). The cited medium was modified from PIKOVSKAIA'S (1948)medium as described by BAxIK and DEY (1982).

Isolation and identification of phosphate solubilizers

16 aluminium phosphate solubilizing microorganisms - 5 bacteria, 5 actinomycetes and 6fungi - were isolated from modified l'IKOVSKAIA'S sucrose-aluminium phosphate agar plates.The isolates were purified by repeated plating and maintained on nutrient agar. The organismswere identified up to generic level according to BERGEY'S Manual (1974) for bacteria, W AKSMAX(1967) for aetinomyeetes, and GILMAX (1957), using Czapek's solution agar (TUITE 1969) for fungi.Bacteria varying from usual morphology were identified up to specific level with the help of Com­monwealth Mycological Institute, Kew, Surrey, U.K.

Determination of phosphate solubilizing power

Phosphate solubilizing power of the isolates, from AlP04 and Ca3(P04)2' were determined inmodified PIKOVSKAIA'S and PIKOVSKAIA'S (1948) broth, as mentioned ealier, by estimatingsoluble phosphorus in 15 ml of the above media, containing 15 mg of insoluble phosphorus and0.15 g of sucrose, after incubating a loopful of culture for bacteria and spores for actinomycetesand fungi in culture tubes. The procedures were the same as described by BANIK and DEY (1981).

Characterization and estimation of organic acids produced

The centrifuged supernatant liquid, obtained from each culture tube after 10 days' incubationwas concentrated in vacuum and analysed paper chromatographically for thermostable andthermolabile organic acids separately (HELD and LEDERER 1951), according to the procedureadopted by BAKIK and DEY (1981).

Results

From Table 1 it is evident that :3.61 per cent of total bacterial and actinomycetepopulation and 1 per cent of total fungal population could thrive on AIP04 as a solesource of phosphorus.

Table 1. Number of AIP04 solubilizers in total soil mieropopulation (x 104)

Organisms Total AIP04 % of AIP04solubilizers

Bacteria and 940 34 3.61ActinomycetesFungi 100 1 1.00

Alluvial Soil Microorganisms 439

Tnhlo~. Pho~phfttr'~oll1lJili7Jin~ power of microorganisms, isolated in sucrose aluminium phosphate

agar plates from alluvial soil

Organisms isolated

Code as Identified as

P-solubilized in,ug/15 mg insoluble P/O.15 gsucrose added(average of duplicate sets)

AlP04

Incubation in days

Meanfortwophos­phates

AAB1 Bacillus sp. 27.5 42.0 34.8AABz Bacillus coagulans 87.0 91.0 89.0AAB 3 Hacillus subtilis 12.0 11.0 11.5AAB 4 Bacillus megatherium 13.0 35.0 24.0AAB, Bacillus sp, 0.0 3.5 1.8AAS1 Streptomyces sp. 21.5 27.5 24.5AASz Streptomyces sp. 0.0 9.5 4.8AAS3 Streptomyces sp, 10.0 25.0 17.5AAS4 Streptomyces sp. 8.0 19.5 13.8AAS, Streptomyces sp. 0.0 0.0 0.0AAF1 Unidentified 0.0 8.0 4.0AAFz Unidentified 1.0 8.0 4.5AAF3 Chaetomium sp. 3.0 21.0 12.0AAF4 Chaetomium sp. 0.0 7.0 3.5AAF, Chaetomium sp. 4.0 6.5 5.3AAF6 Penicillium sp. 27.3 50.0 38.7Mean 13.4 22.8 18.1

Mean for Bacillus (AAB1-AABs) 32.2Mean for Streptomyces (AAS1-AAS,) 12.1Mean for Chaetomium (AAF3-AAFs) 6.9Mean for Penicillium (AAF6) 38.7Mean for Unidentified (AAF1-AAFz) 4.3

1.8 17.03.3 7.73.8 5.40.0 19.40.9 2.6

0.0 0.01.5 6.00.0 0.00.0 0.0

10.0 0.03.5 8.06.0 0.04.0 0.00.0 0.09.5 2.03.0 0.00.5 0.05.5 0.00.0 3.06.5 7.50.0 0.03.1 1.7

7 10 Mean 7 10 Mean

0.0 17.43.8 46.40.0 5.80.0 12.05.0 3.45.8 15.23.0 3.92.0 9.80.0 6.95.8 2.91.5 2.80.3 2.42.8 7.41.5 2.57.0 6.20.0 19.42.4 10.3

LSD at 5 % LSD at 1 %For phosphate (P)

organism (0)Interaction (P X 0) two P at same 0Interaction (P X 0) two 0 at same or different PInteraction (P X I) two I at same PInteraction (P X I) two P at same or different I

5.67.5

10.612.1

3.59.2

28.310.815.326.2

5.039.4

From Table 2 it is observed that all the 5 bacterial isolates were of the genusBacillus - AAB1 to AABs, while the five actinomycetes were of the genus Strepto­myce.s - AAS1 to AASs' and of the 6 fungi, one of the genus Penicillium - AAF 6'

3 of the genus Chaetomium - AAF 3 to AAF s and 2 unidentified ones - AAF1 andAAF 2 . Among the bacilli, morphological variations were observed with Bacilluscoaqulams (B-7659) - AAB2, B. subtilis (B-7660) - AAB3 and B. megatherium(B-7761) - AAB4 ) .

All the isolates, with the exception of Bacillus sp. - AABs, Streptomyces sp. ­AASs and Chaetomium sp. - AAFs were more efficient in solubilizing Ca3(P04)2

than AIP04, despite their isolation in AlP04 agar plates. As a matter of fact B. sub­tili8 (B-7760) - AAB3, B. megatherium (B-7661) - AAB4, Streptomqcee sp. - AAS4,

and Penicillium sp. -AAF6 were unable to accumulate soluble phosphorus in de-

29 Zbl. Mikrobiol., Ed. 138

440 S. BANIK and B. K. DEY

Table 3. Organic acid produced in sucrose-calcium phosphate broth after ten days' incubation bythe microorganisms, isolated in sucrose·aluminium phosphate agar plates from alluvial soil

Organisms

Coded as

AAB1AAB2

AAB3

AAB4

AAB5AAS 1

AAS 2AAS 3

AAS4

AAS 5

AAF1

AAF2AAF3

AAF4

AAF5

AAF6

Identified as

Bacillus sp.Bacillns coagulansBacillus subtiii»Bacillus megatherium

Bacillus sp.Streptomyces sp.Streptomyces sp.Streptomyces sp.

Streptomyces sp.Streptomyces sp.UnidentifiedUnidentifiedChaetomium sp.Chaetomium sp.Chaetomium sp.Penicillium sp.

Organic acids produced

Identified as

Oxalic2-ketogluconicOxalicOxalicMaleicOxalicOxalicOxalicOxalicMalicOxalicOxalicOxalicOxalicOxalicOxalicOxalicOxalic

Amount inmgjO.15 gsucroseadded

Trace2.850Trace2.7751.3503.038Trace0.7501.1631.350Trace3.0383.6750.750Trace1.1252.925Trace

tectable amounts in AlP04 broth, despite their normal growth. On the other hand,Streptomyces sp. - AAS5 behaved similarly in Caa(P04) broth. In general, solubili­zation of Caa(P04h was increased with the incubation period with the exception ofB. subtilie (B-7660) - AABa, where a little decrease was recorded after ten days' ascompared to 7 days' incubation, attributable to the higher role of consumption. Onthe other hand, the situation was different with AlP04 solubilization by the isolatedmicroflora. Only B. coaqulans (B-7659) - AAB2, Streptomyces sp. - AASl' Chaeto­mium sp. - AAS4 and AAS5 solubilized higher amounts after 10 days' than after7 days' incubation. The highest amount of Caa(P04 )2 solubilization was obtained withB. coaqulans (B-7659) - AAB2, followed by Penicillium sp. - AAF6' Bacillus sp. ­AABl' Streptomyces sp. - AAS1 , and B. meqaiherium. (B-766l) - AAB4, while forAlP04 the highest amount of solubilization was recorded with Chaetomium Hp. ­AAF5' followed by Streptomyces sp. - AAS1 and AAS5, Bacillus sp. - AAB6, andB. coaqulans (B-7659) - AAB 2• On an average, when the members of the genera areregarded together, the solubilization from Caa(P04)2 was in the order of Penicillium,Bacillus, Streptomyces, and Chaetomium and from AlP04 in the order Chaeiomium,Streptomyces, Bacillus, and Penicillinm.

In Table 3 it is evident that most of the isolated microorganisms produced oxalicacid. B. megatherinm (B-7661) - AAB4 produced maleic and Streptomyces sp. -AASaproduced malic acid in addition to oxalic acid. 2-ketogluconic acid was producedby B. coagulans (B-7659) - AAB2 only. The isolates, producing 2.ketogluconic acidand maleic acid or malic acid together with oxalic acid, were found to be efficient insolubilizing insoluble inorganic phosphorus. Moreover, the isolates accumulatinglittle quantity of oxalic acid only were found to be efficient solubilizer of Caa(P04)z,

too.

Alluvial Soil Micro organism s 441

Discussion

Peru sal of the results reveal th at aluminium phosphate wluLa;zer cO I\~t, i tut()donly a lit tle part of the total soil microbial population. Although the organisms wereisolat ed from the growth on AIP04 agar , they were more efficient in solubilizingCaa(P04)2' This indi cates that the phosphate solubilizers of alluvial soil were prin­cipally Ca3(P04)2 solubilizers , which exte nded t heir act ivity on other insolubl e ph os­phates only when the availability of Ca3(P0 4)2 became limiting because of t he ty­pical soil characte r. Sustaining earlier reports (BA:NIK and DEY 1981) all t he phos­phate-solubilizing isolates in air-dried soil condit ion were found to be spore-formers .The reason for morphological vari ation in Bacillus coaqulans (B-7659) - AAB2,

B i subtilis (B-7660) - AAB3, and B. meqtuheriusn. (B-7661) - AAB4 might be a fun c­ti on of the present AI+3 ion in th e medium, but awaits fur th er investigation. Despitebeing scant in number , Penicillium showed high Ca3(P04)2 solubilizing potentiality.The impediment to AIP04 solubilizat ion by the trace quantity oxalic acid producersmay be ascribed to the presence offree CaC0 3in broth, deliberately added to maintainthe pH against the hazard of acid production due to partial hydrolysis of AIP04,especially during autoclaving, as t.he first attacking sit e of organic acid was supposedto be carbonate instead of phosphate (ANAND and JHA 1968). Moreover, interactionbetween CaC03 and AIP04 may produce some toxic products during autoclaving,which may restrict growth of inoculated microorganism in broth medium , if notsuppressed growt h in agar medium. However , we are searching for a mor e suitablemedium. Inhibitory effect of free AI+3 ion , accumulated as a consequence of par t ialhydrolysis as mentioned earlier , possibly to a toxic level (ZAJIC 1967), might alsohave played an important role against solubilizat ion from AIP04 and in th e morpho­logical vari ation of th e bacilli. Pr oduction of oxalic acid by the organi sms AABs,AAS2 , AAS3, AAF1 and AAF2 were indeed responsible for utiliz ation of released so­lubl e phos phate in the later stage of growth, which might be one of the causes ofimpediment to accumulate soluble phosphorus in the broth at that stage. However,th e exact cause was not clearl y understood and awai ts fur th er resea rch.

The sole mechanistic pathway of insoluble inorgani c phosphate solubilizat ion bythe microorgani sms has been proposed to be organic acid produ ction (JOHNSTON1954, BANIK and DEY 1981). The organic acids solubi lized either by libera ti on offree proton or by forming chelated compound with Ca+2. 2-ketogluconi c and oxalicacids are well known for the a bove-mentioned dual role (DUFF and WEBLEY 1959).Detection of a little amount of free oxalic acid in the culture broth of microorganisms,solubilizing phosphate with high efficiency , may be at tr ibuted to its formation ofchelates (JOHNSTON 1956). Th e efficiency of releasing insolubl e phosphorus by malicand maleic acids might be due to th eir relatively high er dissociation value (STRL'THERSand SIELING 1950). From the present investigation it is evident that the organism,producing 2-ketogluconic acid, had the highest efficiency in solubilizing insolublephosphate, especially Ca3(P 0 4)2' Th e amount of organic acids accumulat ion in t hegro wth medium was not always indicat ive of the efficiency of phosphate solubili­zation, which was ampl y evidenced by t he isolates accumul ating a lit tle quantityof oxalic acid, possessing high efficiency of Ca3(P0 4h solubilization .

A c kn o wl ed g e m en t

Th e authors thanks are d ue to Council of Scientif ic and Indu st rial Research, India , for pro­viding th e necessa ry financial facili t ies.

442 S. BANIK and B. K. DEY, Alluvial Soil Meroorganisms

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Eingegangen am 20. 7. 1982.

Authors' addresses:

Dr. S. BANIK, Central Marine fisheries Research Institute, P. B. No. 1912, Cochirv - 682018,India; Dr. B. K. DEY, Department of Agricultural Chemistry and Soil Science,Bidhan ChandraKrishi Viswa Vidyalaya, Kalyani 741235, India.