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Phosphate-Solubilizing Potentiality of the Microorganisms Capable of Utilizing Aluminium Phosphate as a Sole Phosphate Source
zu, Mik robi ol.
13 B (1!l83), 17 ·- 23
[Uni ver sit y College of Agri eu lt u t"·, l\1iero biology La bo ra t o ry , Calcu tt a Un iversity, Ca lcutta 7000 19, an d D ep artmen t of Agric ultural Chemistry an d Soil Science, B idhan Ch an d ra Kri ahi Viswa V id ya laya, K al yan i 74 1235, I ndia]
Phosphate-Solubilizing Potenti ality of t he Microorganisms Capable of Utilizing Aluminium Phosphat e as a Sole Phosphate Source S, B ANI K and B, K.
D EY
Summary Eight b acteria, each of t he gen u s Bacillu s, two actino rnycet es, each of t he genus Streptomyces, an d six fungi, one eaeh of the gen u s P enicilli um and Chaetomium an d four of the genus Aspergill u s , were isolated on Al'POj- aucrose agar fro m a t ypical Indian later it ic soil (T ypic Ochragualf) . All of t hem wer e ca pa ble of solu bili zing Ca. (P 0 4 ) 2 to a hi gh er deg ree t han AlP 0 4 • B acill u s subt ilis (B· 7( 55), LAB4 , B acillu s sp ., L ABs' P en icill ium sp., LAF 2 ' an d A sp ergillus s pp., L AF . an d L AF 4 , were solubilizing Ca. (P 0 4 h ve ry eff ieiently, but AIP0 4 t o a lesser degree. Ba cillus sp p. , LA B !, LAB 2 , LAB s, LAB B and LA B" Chaetomium n iqri color, LAF!, and A sp ergillus s pp., LAF s and LAF 6 , were unable t o b ring detect able a mou nts of soluble ph osphoru s to solut ion fr om AIP 0 4• E xcep t Uhaetomium , all t he other orga n isms p rod u ced free ali phat ic o rga n ic ac id in de · t ect a ble a mou nts. T he o rganic adds p ro du ced wer e oxa lic , su ccin ic, cit ric, a n d 2-keto gluconic acid . 2-Keto glu conic acid , sing ly an d in eom bin at ion with succ in ic 01' r-it r ic ucid , acc ounted for higher solubilization. Am ount of fr ee organie ac ids in t he growth medium was not direct ly corre lat ed wit h phosp hat e solubi lizatio n .
Zusammenfassung Ach t B akterien de l' Gatt ung Bacillus, zwei Aktinomyzeten der Gattu ng S trep tom yces u nd seehs P ilze , ei ne Pe nicill iu m- , eine Ohaetomium- und v ie r Aspergillt,s· A rten, wu rden a u f A IP0 4 Sukr ose-Agar a us eine m t ypischen in d ischen Lateri t (T'y piscbe r Och ragu alf] isolier t. Alle di ese Mikroorganismon waren fah ig, Ca.(P0 4 ) 2 in eine m hoh eren Ma Be zu losen als AIP0 4 • B acillus su btilis (13-7655 ) L AB 4 , B acill us sp, LAF 2 und Aspergillus s pp, L AF . und LAF 4 lost en Ca.( P 0 4 h sehr wirk sam , AIP0 4 jed och in geringem Mal3e. B acillu s sp p. LA B!, LAB 2 , L AB s, L AB 6 und LA B" Chaetomiu m niqri color L AF 1 und Aspergillu s spp. L AF s u n d L AF 6 waren ni ch t in del' L age, nennens wert e Men gen an Phosphor au s AIP0 4 zu losen. Aulser Chaetom ium bildeten aIle an de ren Organismen frei e alip hatische organische Sauren in naehw ei sbaren Mengen: Bernst einun d Zit ronens au re sowi e 2-Ket o·Glukonsau re, VOl' allem 2-Keto· Glu kon sa ur e, allein oder in Vel" bi n du ng m it Bernstein- od er Zit ro nensaure, d urfte die L oslichk eit erhohen, Die Konzent r at ion del' freien orga n ische n Saul'en im Wachs t u msm ediu m stand ni cht in d ire-k t er Bezi ehung zur Phosphatloslichkeit .
Tr opical soil ph osph at es are mostly interlocked in insoluble inorganic form s 1973). Among insoluble inorganic soil ph osph at es ferri c(III) and alum inium (III) phosphates are dominating in acid soils (pH less t han 6.5) and t ricalcium ph osphate in neutral to alkaline soils (pH above 6.5) (R USSELL 197:3). Diverse genera of soil population , belonging to ba cteria, act inomycete s, and moulds, hav e been reo ported t o be active in solubilizing insoluble inorgani c ph osph at es with high efficiency (SPER B E R 1958 ; H An l AN 1975 ; BANIK and DE Y 1981). Among them an ap preciable number can be t hought t o t hrive on AlP 0 4, espec ially in acid soils. El aboration of orga nic acid" by t he responsible organisms is th e major mean of mobi-
( R USSEL L
2
Zbl. nli kro bioJ., TId. 138
18
S. B ANIK and B . K. D EY
lizing insoluble phosph at es (.JO HNS'l'ON 1954-). Th e present investigation is a st udy of characterization of t he microflora , utilizing AI PO~ , present in a lateritic soil (Typic Ochragualf, R U DR A 1975), their phosph ate-solubil izing pot entialit y , and pr odu ct ion of aliphatic organic acids during solub ilizat ion in in. vitro exp erim ent s.
Materials and Methods T he soil sa m ple was colle cte d fro m Sevabharati Experimental Station of Calcu t t a Uni versity, K apgari , Di strict Mid nupo re, West Bengal, India. The so il wa s ide ntified as Typic Och ra gu al f (Ru DRA 1975) a cco r ding to t he Seventh Approximation. Air- d ried 2 mm sieved soil sam ples were u sed in the present st u d y. T ot al phosphoru s and differ ent fra ct ions of soil phosph at es, present in the soil, were est im ate d in ac cordance with J ACKSON (1967) and CIIA~W and JA CKSON (1957) , resp ecti vely , and tabulat e d in T a ble 1. Table 1. Different fra cti on s of phosphorus, pr esen t in the later iti c soil used Soil L at eri ti c
Total 1'1) 29.80
Available l' 0.70
F er ric P
Culcium P.
11.0
3.50
Total org ani c
p
5.80 Occluded alum inium l' 0.38
Water- solu ble p 0.08
Occlud ed ferric l'
To ta l in o rgan ic P
0.00
24.96
Alumini um P 10. 00
% of Alum inium P in total soil P 34.90
1) expressed in mg/ l00 g a ir-d ry soil
Enumerati on of micr oorganisms T ot al b act eria were enu me r-ated i n B UNT an d R OVIRA' S (1955) agar, uct.inom ycet es in JENSE,, 'S (1930) agar , fu ng i in MARTI~ 'S (1950) ro se b engal streptomycin aga r a n d alu m iniu m phosphate-sol ubilizing org anism s (bacteria , actino m ycet es , an d fungi) in modified (by t he au t hors ) P IKOVSKAIA'S (1948) agar media , resp ectively, following serial di lution t echnique. T he lat t er m edium was modified b y u sing 4.0 g of AIP0 4 in pl ace of 5.0 g of Cu. (P0 4 b cont ainin g equ ivalent amount of phosphorus , an d 0.5 g of Ca CO. pel' lit re m ed ium t o maintain pH at (i.8 .
Isolation and identification of phosphate solubilizers Sixteen alu m in iu m phosphate -solubi lizi ng m icroorgani sm s - eight b act eri a , two a ct inomyeetes, and six fungi - wer e isolated fro m modifie d Piko vsk ai a's su crose-Al PO, agar pl at es. The isolates were purified by re peat ed plating an d coded as L AB , LAS, and LAF with suffix numbe rs for b act eria , strept omycetes (actinom ycetes), an d funti, respectively. The cul t u res were main tained on nu t r ien t ag ar medi u m. These were identified u p to ge ne ric level ac cor din g to BERGEY'S manu al (1974) for bacteria, WAKSMAN (1967 ) for actino mycetes an d GI LMAN (1957 ), u sin g CZAPEK'S solut ion agar (T UITE 1969), for fungi. B acteria sho wing va riations in mo rp hology were id entified u p to spe cific level wit h t he help of Com monwealth Mycological I nsti tute, K ew, Surrey, U .K ., an d t he fu ngi Chaetomiu rn wit h t he help of Mycological L abo rato r y, D ep artm en t of Bot an y , Univer sity of Calcut ta.
Det erm inat ion of phosph ate-solubili zing power Phosphate-solubilizin g power of the isol ates from AIP0 4 and Ca. (1'04) 2 were deter m in ed in modified Pikovsk aia' s and l'iko vsk aia 's (1948) broth, as m ent ioned earlier, by est im ating soluble phosphorus in 15 ml of the ab o ve medi a, cont ain ing 15 mg of insoluble p hos phoru s and 0.15 g of sucrose, after in cubat ing 0. 1 m1 of hea vy su spension of t he growt h fo r bacter ia an d spores for actin omycetes an d fu ngi in t h e cultu re t ubes . The pro cedures were t he same as descri b ed by BANIK and D EY (1981).
Pho ~ p h Ht P- ~olllb il i z i llg
Pot ent ia lit y of t he
:\li e l'O o rgalli ~ lIl s
IH
Characterization an d est imat ion of organic acids produced T he centrifuged su pe rnatan t li quid. obta ine d from ea ch cult u re t u be . containi ng s ur-ro se Ca 3(P 0 4h b rot h ( P IKOVSKAIA 1948). a fter 10 d a ys' incu b at ion was conce nt ra te d in Va('UUIII a nd analysed pa pe r ch romat ographically for t he rm ost a ble an d thermolabile a lip h at ic org ani c a cids separa te ly (REID and L E DE R E R 1951).
Results F ra ctionation of soil phosph oru s indicate s t hat th e major amount of ph osph orus is locked in insolubl e inorga nic phosph atic form s, of which 34.90 % (Table 1) is contributed by th e different forms of aluminium phosph ate. Ta ble 2 shows that 15.8 % of th e to tal ba cteria and actinornycetes and ] A % of the tot al fungi could thrive and solubilize AIP0 4 . Table 2. Number of AIP0 4 solubilizers in t otal soilmict'Oorganism s ( x lOt)
o r'ganisms
Tot al
AIP(\
% of AlPOt solubilizers
B uct eri a a nd Actinomycetes F u ng i
(53!)
99.5
15.S
140
2.0
1.4
F rom Table :3 it can bc seen th at all t he eight bacteria l isolates were of t he genus B acillus LAB! to LABs, while th e two actinomycet es were of t he genus Strep tomuces LASt and LAS2 , and of six fungi, one each of t he genus P enicillium and Chaetomium and four of the genus A spergillus. Th e bacilli l . AB t , LAB3, L AB4, LAB 7, and I.. ABs, deviating from usual morph ology, were identifi ed up t o specific level and were found to be morphological variants of Bacillus subtilis. Ohaetomium. sp., found for t he first ti me as ph osph ate solubi lizer, was also identified up to specific level as (lhaeiomium
niqricolor.
Th e above organisms showed high efficiency in solubilizing Ca3( P O.1)2 despite t heir isolat ion in AIP0 4 aga r plat es. As a matter of fact , fiv e B acill us spp ., LABI , LAB 2 , LAB 5 , LAB 6 , and LAB" Chaetomi um. niqricolor I. AF t , and A spergillus spp . LAI!\ and I.. AF 6 were unable t o accumulate soluble ph osph oru s in detectable amounts in broth . The efficien cy of solubilizing AIP0 4 were in th e order Penicillium LA]'2' Aspergillus LAF3 , LAF 4 , Ba cillu s subtiii« LAB 4 , Str eptomyces LAS 2 , Bacillus subtilis J.. AB3 , S treptomyces LAS!, an d B acillus subtilie LABs. Overall efficiency of the genera were in the order P enicill iu m , A spergillus, Streptomyc es, B acillus, and Chaetomium . The amount of AIP0 4 solubilization was essentially increased wit h the incubation period. As regards Ca3( P 0 4)2 solubilizatio n, an entirely different p icture was obtained . The bacilli showed higher solubilization afte r 7 days th an after 10 days, exce pt for LABs, while th e reverse was t rue for t he fungi, except for A spergillus LAF 3 . St reptomyces LAS2 behaved like t he fun gi, while LAS! like t he ba cilli in t his respect . On an average , as a genus, P enicillium solubilized the highest amount of Ca 3( P 0 4)2' followed by A spergillus, Ba cillus, Str eptomyces, and Chaeiomium. Individu ally , A sperqill us LAI!~4 was found to solubilize t he great est amount of Ca3(P 0 4)2' while P enicillium I.AFz' B acillus LAB5 and LAB<\., A spergillus L AF 3 , B adllus LABs, A spergillus LAFr, and L AF 5 followed. In general, t he solu bilization was greate r from Ca3 (P 0 4 ) 2 t han from AlP 0 4 • 2'
20
S.
BANIK
and
B.
K.
DEY
Table 3. Phosphate-solubilizing power of microorganisms, isolated in sucrose aluminium phosphate agar plates Organisms isolated Coded as
Identified as
Mean for two phos phates
P-solubilized in Jlg/15 mg insoluble P/O.15 g sucrose consumed (average of duplicate sets) Ca 3 (P 0 4h
AIP0 4
Incubation in days
7
10
Mean
10
7
Mean
LAB l
Bacillus subtili« B-7653
28.3
24.5
26.4
0
0
0
13.2
LAB 2
Bacillus
31.0
24.0
27.5
0
0
0
13.8
LAB 3
Bacillus subtili« B-7654
41.5
7.0
24.3
0
7.4
3.7
14.0
LAB4
Bacillus subtilis B-7655
133.0
118.0
125.5
39.0
21.3
73.4
LAB 5
Bacillus
154.0
135.8
144.9
0
0
0
72.5
LAB 6 LAB 7
Bacillus
23.0 20.3
16.5 11.0
19.8 15.7
0
0
0
0
0 0
9.9 7.9
3.0
0
1.5
52.7
0
5.0
2.5
16.0
Bacillus subtilis B-7657
3.5
LAB g
Bacillus subtilis B-7658
91.5
116.0
103.8
LAS l
Streptomyces
30.0
29.0
29.5
LAS 2
Streptomyces
19.0
28.5
23.8
7.5
17.5
12.5
18.2
LAF l LAF 2
Chaetomium nigricolor Penicillium Aspergillus Aspergillus Aspergillus Aspergillus
7.0 151.0 129.5 126.0 36.5 73.5
21.0 180.5 119.5 208.0 66.0 95.0
14.0 165.8 124.5 167.0 51.3 84.3
0 81.5
0 70.8
7.0 118.3
24.0 6.5
0 60.0 56.5
0 0
41.0 0 0
40.3 23.8 0 0
95.4 25.7 42.2
68.4
75.0
71.8
7.9
14.2
11.0
41.4
LAF 3 LAF4 LAF 5 LAF 6
Mean
Mean Mean Mean Mean Mean
for for for for for
Bacillus (LAB l - LAB g) Streptomyces (LAS l - LAS 2 ) Chaetomium (LAF l ) Penicillium (LAF 2) Aspergillus (LAF 3-LAF6 )
For phosphate (P) For Organism (0) Interaction (P x 0) - two P at same 0 Interaction (P x 0) - two 0 at same or different P Interaction (P x 0 x I) - two I at same P and 0 Interaction (P x 0 x I) - two 0 at same P and I Interaction (P x 0 x I) - two P at same 0 and I
B-7653 to B-7658 are CMI code numbers.
82.4
Mean
Mean
Mean for two
61.0 26.7 14.0 Hi5.8 106.8
3.3 7.5 0 70.8 16.0
32.2 17.1 7.0 118.3 61.4
CD at 5%
CD at 1%
25.5 16.7 23.7 28.1 32.0 32.7 35.7
128.2 24.2 34.3 60.5 46.2 47.4 101.2
Phosphate-solubilizing Pnt eut iu lit y of t hr- :\li('l'oorganisluR
21
'fable 4. Organic acids, produced in sucrose-culr-ium phosphate brot h uf'tr-r ten days' incubation by the microorganisms, isolated in sucrose-aluminium phosphate agar plates Organisms
Organic aeids produced
Coded as
Identified as
Identified as
Amount in mg/0.15 g sucrose consumed
LAB l
B acillus subtilis B-7653
Oxalic Succinic
2.775 0.938
LAB 2
Bacillns
Oxalic
UOO
LAB a
Bacillns snbtilis B-7fi54
Oxalic
2.513
LAB4
Bacill us subtilis B-7fi55
2-ketoglueonic Succinic
3.375 4.538
LAB 5
Bacillns
2-ketogluconie Succinic
2.400 3.488
LAB G
BacilluH
Oxalic
1.650
LAB)
Bacillns subtili« B-7(j57
Oxalic
3.300
LABs
Bacillus subtilis B·7658
2-ketogluconic
3.300
LAS l
Streptomyces
Oxalic
1.800
LAS 2
Streptomyces
Oxalic
2.100
LAF l
Chaetomium. nigricolor
LAF2
Pen'icillium
Citric' 2-ketogluconie
5.250 1.500
LA Fa
Aspergillus
Citric 2-ketogluconic
4.388 4.125
LAF4
Asperqillu»
2-ketogluconic
3.150
LAl<\ LAF G
A sperqilius
2-ketogluconic
2.175
Asperqilius
2-ketogluconic
4.200
- indicates organic acid not detected. B-7fi53 to B-7fi58 are CMI code numbers.
Microorganisms produced oxalic, succinic, citric, and 2.ketogluconic acid in sucrose-calcium phosphate broth (Table 4). Although the amount of free organic acid was not correlated with phosphate solubilization, the efficient solubilizers produced 2-ketogluconic acid either singly or in combination with succinic or citric acid. No aliphatic organie acid was produced by Chaeiomium niqricolor in detectable amounts.
Discussion Perusal of the results reveal aluminium phosphate solubilizers to be fewer in population in comparison to Ca3(P0 4)2 solubilizers, and although the organisms were isolated on AIP0 1 , they were more efficient in solubilizing Ca3(PO j )2 ' The indicates that the phosphate solubilizers in the lateritic soil studied were principally Ca3(P() 4)2 solubilizers, They extended their ability to other insoluble phosphate only when its
22
S. BANIK and B. K. DEY
availability became limited. Isolation of the spore formers indicated the prevailing stress condition (ALEXANDER 1977) in the soil studied, especially in terms of texture, pH, organic matter, and nitrogen content (BANIK and DEY 1981). The reason for the morphological variation of some bacilli remained obscure and awaits further investigation. Despite being scant in number, the fungi Penicillium showed the highest solubilizing ability, most probably by virtue of their production of higher amounts of aliphatic organic acids, as suggested by earlier workers (DUFF et al. 1963 ; JOHNsrox 1954). Sustaining the findings by ANAND and JHA (1968), occasional species of Bacillus brought appreciable amounts of soluble phosphorus from insoluble phosphatic sources. The newly found Chaetomium nigricolor was not so efficient in this respect, presumably due to its inability of producing aliphatic organic acids in considerable amounts. Although no generalization in terms of efficiency can be drawn for a genus or species as regards production of a particular organic acid, it was well established by the previous work (BANIK and DEY 1981) and the present data that the organisms, producing 2-ketogluconic acid in combination with succinic or citric, were very efficient in solubilizing insoluble inorganic phosphates. The impediment to AIP0 4 solubilization in broth may be ascribed to the presence of CaC0 3, deliberately added to the broth to maintain the pH, as the first attacking site of the organic acids was supposed to be carbonate instead of phosphate (ANAND and JHA 1968). Nevertheless, the presence of available phosphate in the medium as Ca(H 2P0 4)+ and Ca(HP0 4 ) by the action of free H+, liberated due to partial hydrolysis of AIP0 4 during autoclaving, might have restrained the organisms from utilizing the insoluble phosphate through solubilizing at least in the earlier stage of growth. Inhibitory effect of the consequent Al+3 ion, possibly to a toxic level, to the metabolism of the organisms (ZAJIC 1967) might have played an important role on the variation in morphology of the bacteria. Increase in solubilization of AIP0 4 with the incubation time may be ascribed to the activity of the resistant forms. From the foregoing discussion it becomes clear that tropical soil phosphate solubilizers are essentially Ca3(P04}z solubilizers: they attack other insoluble sources when the earlier substrate is unavailable. However, the exact role of such microorganisms may go beyond expectation in natural soil condition. So, more extensive research in the field will be needed for better understanding of such microorganism - soil relationship and their beneficial role to crop plants. Acknowledgement vVe are grateful to Council of Scientific and Industrial Research, India, for financial help.
References ALEXANDER, M.: Introduction to soil Microbiology. John Wiley & Sons, Inc., New York and London 1977. ANAND, N., and JHA, K. K.: Solubilization of rock phosphate by microorganisms isolated from Bihar soils. J. Gen. Appl. Microbiol. 14 (1968),89-95. BANIK, S., and DEY, B. K.: Phosphate-solubilizing microorganisms of a lateritic soil. 1. Solubilization of inorganic phosphates and production of organic acids by microorganisms, isolated in sucrose calcium phosphate agar plates. Zbl. Bakt. II 136 (1981),478-486. BERGEY'S Manual of Determinative Bacteriology, 8th edn. (BUCHANAN, R. E., and GIBBONS, N. E., eds.), Baltimore 1974. BL:NT, J. S., and ROVIRA, A. D.: Microbiological studies of some subanturr-t.ie soils. J. Soil Sci. 6(1955), 119~128. CHANG, S. C., and JACKSON, M. L.: Fractionation of soil phosphorus. Soil Sci. 84 (1957), 133-144 DUFF, R. E., WEBLEY, D. M., and SCOTT, R. 0.: Solubilization of minerals and related minerals by 2-ketoglueonic acid producing bacteria. Soil Sci. 95 (1963), 105-114.
Phosphate-solubilizing Potentiality of t hr- Microorganisms
2~
GILMAN, J. C.: A manual of soil fungi. 2nd rev. edn. Iowa State College Press 1957. HAYMAN, D. S.: Phosphorus cycling by soil microorganisms and plant roots. Soil Miembiology (WALKER, N., ed.). Butterworths, London 1975, 67 -92. JACKSON, M. L.: Soil chemical analysis. Prentiee-Hall of India Pvt. Ltd., New Delhi 1967. JENSEN, H. L.: Azotobacteriaceae. Bact. Rev. 189 (1930), 195-2.14. JOHNSTON, H. W.: The solubilization of insoluble phosphate. II. A quantitative and comparative study of the action of seleeted aliphatic acids on tricalcium phosphate. N.Z.J.Sei. Teehn. 36 (1954),49-55. MARTIN, J. P.: Use of acid, rosobengal and streptomycin in the plate method for estimating soil fungi. Soil Sci. 69 (1950), 215 - 232. PIKOVSKAIA, R. 1.: Mobilization of phosphates in soil in «onnection with vital aetivity of some microbial species. Mikrobiologiya 17 (1948), 362-370. REID, R. L., and LEDERER, M.: Separation and estimation of sat.urod Cz -C 7 fatty acids by paper partition chromatography. Biochem. J. 50 (1951), 60-67. RlJDRA, P.: Characteristics and genesis of some of the lateritie soils of West Bengal, leading to their elassifieation and land use planning. Ph.D. Thesis, Calcuttu University, Calcutta, India (1975). RUSSELL, E. VV.: Soil condition and plant growth. 10th edn. New York 1973. SPERBER, J. 1.: The incidence of apatite solubilizing organisms in the rhizosphere and soil. Aust. J. Agrie. Res. 9 (1958),778-787. TlJITE, J.: Plant Pathological Methods - Fungi and Bacteria, Burgess Publishing Co., Minneapolis 1959, 239. WAKSMAN, S. A.: The Aetinomyeetes. Ronald Press Co., New York 1967, 280. ZAJIC, J. E.: Microbiol Biogeochemistry, Academic Press. New York and London 1967, 345. Eingegangen am 8. 2. 1982. Authors' addresses: Dr. S. BANIK, University College of Agrieulture, Microbiologieal Laboratory, Calcutta University, Calcutta 700019, and Dr. B. K. DEY, Department of Agricultural Chemistry and Soil Scienee, Faeulty of Agriculture, Bidhan Chandra Krishi Viswa Vidyalaya, Kalyani, Nadia, Pin 741235, West Bengal, India.
13 B (1!l83), 17 ·- 23
[Uni ver sit y College of Agri eu lt u t"·, l\1iero biology La bo ra t o ry , Calcu tt a Un iversity, Ca lcutta 7000 19, an d D ep artmen t of Agric ultural Chemistry an d Soil Science, B idhan Ch an d ra Kri ahi Viswa V id ya laya, K al yan i 74 1235, I ndia]
Phosphate-Solubilizing Potenti ality of t he Microorganisms Capable of Utilizing Aluminium Phosphat e as a Sole Phosphate Source S, B ANI K and B, K.
D EY
Summary Eight b acteria, each of t he gen u s Bacillu s, two actino rnycet es, each of t he genus Streptomyces, an d six fungi, one eaeh of the gen u s P enicilli um and Chaetomium an d four of the genus Aspergill u s , were isolated on Al'POj- aucrose agar fro m a t ypical Indian later it ic soil (T ypic Ochragualf) . All of t hem wer e ca pa ble of solu bili zing Ca. (P 0 4 ) 2 to a hi gh er deg ree t han AlP 0 4 • B acill u s subt ilis (B· 7( 55), LAB4 , B acillu s sp ., L ABs' P en icill ium sp., LAF 2 ' an d A sp ergillus s pp., L AF . an d L AF 4 , were solubilizing Ca. (P 0 4 h ve ry eff ieiently, but AIP0 4 t o a lesser degree. Ba cillus sp p. , LA B !, LAB 2 , LAB s, LAB B and LA B" Chaetomium n iqri color, LAF!, and A sp ergillus s pp., LAF s and LAF 6 , were unable t o b ring detect able a mou nts of soluble ph osphoru s to solut ion fr om AIP 0 4• E xcep t Uhaetomium , all t he other orga n isms p rod u ced free ali phat ic o rga n ic ac id in de · t ect a ble a mou nts. T he o rganic adds p ro du ced wer e oxa lic , su ccin ic, cit ric, a n d 2-keto gluconic acid . 2-Keto glu conic acid , sing ly an d in eom bin at ion with succ in ic 01' r-it r ic ucid , acc ounted for higher solubilization. Am ount of fr ee organie ac ids in t he growth medium was not direct ly corre lat ed wit h phosp hat e solubi lizatio n .
Zusammenfassung Ach t B akterien de l' Gatt ung Bacillus, zwei Aktinomyzeten der Gattu ng S trep tom yces u nd seehs P ilze , ei ne Pe nicill iu m- , eine Ohaetomium- und v ie r Aspergillt,s· A rten, wu rden a u f A IP0 4 Sukr ose-Agar a us eine m t ypischen in d ischen Lateri t (T'y piscbe r Och ragu alf] isolier t. Alle di ese Mikroorganismon waren fah ig, Ca.(P0 4 ) 2 in eine m hoh eren Ma Be zu losen als AIP0 4 • B acillus su btilis (13-7655 ) L AB 4 , B acill us sp, LAF 2 und Aspergillus s pp, L AF . und LAF 4 lost en Ca.( P 0 4 h sehr wirk sam , AIP0 4 jed och in geringem Mal3e. B acillu s sp p. LA B!, LAB 2 , L AB s, L AB 6 und LA B" Chaetomiu m niqri color L AF 1 und Aspergillu s spp. L AF s u n d L AF 6 waren ni ch t in del' L age, nennens wert e Men gen an Phosphor au s AIP0 4 zu losen. Aulser Chaetom ium bildeten aIle an de ren Organismen frei e alip hatische organische Sauren in naehw ei sbaren Mengen: Bernst einun d Zit ronens au re sowi e 2-Ket o·Glukonsau re, VOl' allem 2-Keto· Glu kon sa ur e, allein oder in Vel" bi n du ng m it Bernstein- od er Zit ro nensaure, d urfte die L oslichk eit erhohen, Die Konzent r at ion del' freien orga n ische n Saul'en im Wachs t u msm ediu m stand ni cht in d ire-k t er Bezi ehung zur Phosphatloslichkeit .
Tr opical soil ph osph at es are mostly interlocked in insoluble inorganic form s 1973). Among insoluble inorganic soil ph osph at es ferri c(III) and alum inium (III) phosphates are dominating in acid soils (pH less t han 6.5) and t ricalcium ph osphate in neutral to alkaline soils (pH above 6.5) (R USSELL 197:3). Diverse genera of soil population , belonging to ba cteria, act inomycete s, and moulds, hav e been reo ported t o be active in solubilizing insoluble inorgani c ph osph at es with high efficiency (SPER B E R 1958 ; H An l AN 1975 ; BANIK and DE Y 1981). Among them an ap preciable number can be t hought t o t hrive on AlP 0 4, espec ially in acid soils. El aboration of orga nic acid" by t he responsible organisms is th e major mean of mobi-
( R USSEL L
2
Zbl. nli kro bioJ., TId. 138
18
S. B ANIK and B . K. D EY
lizing insoluble phosph at es (.JO HNS'l'ON 1954-). Th e present investigation is a st udy of characterization of t he microflora , utilizing AI PO~ , present in a lateritic soil (Typic Ochragualf, R U DR A 1975), their phosph ate-solubil izing pot entialit y , and pr odu ct ion of aliphatic organic acids during solub ilizat ion in in. vitro exp erim ent s.
Materials and Methods T he soil sa m ple was colle cte d fro m Sevabharati Experimental Station of Calcu t t a Uni versity, K apgari , Di strict Mid nupo re, West Bengal, India. The so il wa s ide ntified as Typic Och ra gu al f (Ru DRA 1975) a cco r ding to t he Seventh Approximation. Air- d ried 2 mm sieved soil sam ples were u sed in the present st u d y. T ot al phosphoru s and differ ent fra ct ions of soil phosph at es, present in the soil, were est im ate d in ac cordance with J ACKSON (1967) and CIIA~W and JA CKSON (1957) , resp ecti vely , and tabulat e d in T a ble 1. Table 1. Different fra cti on s of phosphorus, pr esen t in the later iti c soil used Soil L at eri ti c
Total 1'1) 29.80
Available l' 0.70
F er ric P
Culcium P.
11.0
3.50
Total org ani c
p
5.80 Occluded alum inium l' 0.38
Water- solu ble p 0.08
Occlud ed ferric l'
To ta l in o rgan ic P
0.00
24.96
Alumini um P 10. 00
% of Alum inium P in total soil P 34.90
1) expressed in mg/ l00 g a ir-d ry soil
Enumerati on of micr oorganisms T ot al b act eria were enu me r-ated i n B UNT an d R OVIRA' S (1955) agar, uct.inom ycet es in JENSE,, 'S (1930) agar , fu ng i in MARTI~ 'S (1950) ro se b engal streptomycin aga r a n d alu m iniu m phosphate-sol ubilizing org anism s (bacteria , actino m ycet es , an d fungi) in modified (by t he au t hors ) P IKOVSKAIA'S (1948) agar media , resp ectively, following serial di lution t echnique. T he lat t er m edium was modified b y u sing 4.0 g of AIP0 4 in pl ace of 5.0 g of Cu. (P0 4 b cont ainin g equ ivalent amount of phosphorus , an d 0.5 g of Ca CO. pel' lit re m ed ium t o maintain pH at (i.8 .
Isolation and identification of phosphate solubilizers Sixteen alu m in iu m phosphate -solubi lizi ng m icroorgani sm s - eight b act eri a , two a ct inomyeetes, and six fungi - wer e isolated fro m modifie d Piko vsk ai a's su crose-Al PO, agar pl at es. The isolates were purified by re peat ed plating an d coded as L AB , LAS, and LAF with suffix numbe rs for b act eria , strept omycetes (actinom ycetes), an d funti, respectively. The cul t u res were main tained on nu t r ien t ag ar medi u m. These were identified u p to ge ne ric level ac cor din g to BERGEY'S manu al (1974) for bacteria, WAKSMAN (1967 ) for actino mycetes an d GI LMAN (1957 ), u sin g CZAPEK'S solut ion agar (T UITE 1969), for fungi. B acteria sho wing va riations in mo rp hology were id entified u p to spe cific level wit h t he help of Com monwealth Mycological I nsti tute, K ew, Surrey, U .K ., an d t he fu ngi Chaetomiu rn wit h t he help of Mycological L abo rato r y, D ep artm en t of Bot an y , Univer sity of Calcut ta.
Det erm inat ion of phosph ate-solubili zing power Phosphate-solubilizin g power of the isol ates from AIP0 4 and Ca. (1'04) 2 were deter m in ed in modified Pikovsk aia' s and l'iko vsk aia 's (1948) broth, as m ent ioned earlier, by est im ating soluble phosphorus in 15 ml of the ab o ve medi a, cont ain ing 15 mg of insoluble p hos phoru s and 0.15 g of sucrose, after in cubat ing 0. 1 m1 of hea vy su spension of t he growt h fo r bacter ia an d spores for actin omycetes an d fu ngi in t h e cultu re t ubes . The pro cedures were t he same as descri b ed by BANIK and D EY (1981).
Pho ~ p h Ht P- ~olllb il i z i llg
Pot ent ia lit y of t he
:\li e l'O o rgalli ~ lIl s
IH
Characterization an d est imat ion of organic acids produced T he centrifuged su pe rnatan t li quid. obta ine d from ea ch cult u re t u be . containi ng s ur-ro se Ca 3(P 0 4h b rot h ( P IKOVSKAIA 1948). a fter 10 d a ys' incu b at ion was conce nt ra te d in Va('UUIII a nd analysed pa pe r ch romat ographically for t he rm ost a ble an d thermolabile a lip h at ic org ani c a cids separa te ly (REID and L E DE R E R 1951).
Results F ra ctionation of soil phosph oru s indicate s t hat th e major amount of ph osph orus is locked in insolubl e inorga nic phosph atic form s, of which 34.90 % (Table 1) is contributed by th e different forms of aluminium phosph ate. Ta ble 2 shows that 15.8 % of th e to tal ba cteria and actinornycetes and ] A % of the tot al fungi could thrive and solubilize AIP0 4 . Table 2. Number of AIP0 4 solubilizers in t otal soilmict'Oorganism s ( x lOt)
o r'ganisms
Tot al
AIP(\
% of AlPOt solubilizers
B uct eri a a nd Actinomycetes F u ng i
(53!)
99.5
15.S
140
2.0
1.4
F rom Table :3 it can bc seen th at all t he eight bacteria l isolates were of t he genus B acillus LAB! to LABs, while th e two actinomycet es were of t he genus Strep tomuces LASt and LAS2 , and of six fungi, one each of t he genus P enicillium and Chaetomium and four of the genus A spergillus. Th e bacilli l . AB t , LAB3, L AB4, LAB 7, and I.. ABs, deviating from usual morph ology, were identifi ed up t o specific level and were found to be morphological variants of Bacillus subtilis. Ohaetomium. sp., found for t he first ti me as ph osph ate solubi lizer, was also identified up to specific level as (lhaeiomium
niqricolor.
Th e above organisms showed high efficiency in solubilizing Ca3( P O.1)2 despite t heir isolat ion in AIP0 4 aga r plat es. As a matter of fact , fiv e B acill us spp ., LABI , LAB 2 , LAB 5 , LAB 6 , and LAB" Chaetomi um. niqricolor I. AF t , and A spergillus spp . LAI!\ and I.. AF 6 were unable t o accumulate soluble ph osph oru s in detectable amounts in broth . The efficien cy of solubilizing AIP0 4 were in th e order Penicillium LA]'2' Aspergillus LAF3 , LAF 4 , Ba cillu s subtiii« LAB 4 , Str eptomyces LAS 2 , Bacillus subtilis J.. AB3 , S treptomyces LAS!, an d B acillus subtilie LABs. Overall efficiency of the genera were in the order P enicill iu m , A spergillus, Streptomyc es, B acillus, and Chaetomium . The amount of AIP0 4 solubilization was essentially increased wit h the incubation period. As regards Ca3( P 0 4)2 solubilizatio n, an entirely different p icture was obtained . The bacilli showed higher solubilization afte r 7 days th an after 10 days, exce pt for LABs, while th e reverse was t rue for t he fungi, except for A spergillus LAF 3 . St reptomyces LAS2 behaved like t he fun gi, while LAS! like t he ba cilli in t his respect . On an average , as a genus, P enicillium solubilized the highest amount of Ca 3( P 0 4)2' followed by A spergillus, Ba cillus, Str eptomyces, and Chaeiomium. Individu ally , A sperqill us LAI!~4 was found to solubilize t he great est amount of Ca3(P 0 4)2' while P enicillium I.AFz' B acillus LAB5 and LAB<\., A spergillus L AF 3 , B adllus LABs, A spergillus LAFr, and L AF 5 followed. In general, t he solu bilization was greate r from Ca3 (P 0 4 ) 2 t han from AlP 0 4 • 2'
20
S.
BANIK
and
B.
K.
DEY
Table 3. Phosphate-solubilizing power of microorganisms, isolated in sucrose aluminium phosphate agar plates Organisms isolated Coded as
Identified as
Mean for two phos phates
P-solubilized in Jlg/15 mg insoluble P/O.15 g sucrose consumed (average of duplicate sets) Ca 3 (P 0 4h
AIP0 4
Incubation in days
7
10
Mean
10
7
Mean
LAB l
Bacillus subtili« B-7653
28.3
24.5
26.4
0
0
0
13.2
LAB 2
Bacillus
31.0
24.0
27.5
0
0
0
13.8
LAB 3
Bacillus subtili« B-7654
41.5
7.0
24.3
0
7.4
3.7
14.0
LAB4
Bacillus subtilis B-7655
133.0
118.0
125.5
39.0
21.3
73.4
LAB 5
Bacillus
154.0
135.8
144.9
0
0
0
72.5
LAB 6 LAB 7
Bacillus
23.0 20.3
16.5 11.0
19.8 15.7
0
0
0
0
0 0
9.9 7.9
3.0
0
1.5
52.7
0
5.0
2.5
16.0
Bacillus subtilis B-7657
3.5
LAB g
Bacillus subtilis B-7658
91.5
116.0
103.8
LAS l
Streptomyces
30.0
29.0
29.5
LAS 2
Streptomyces
19.0
28.5
23.8
7.5
17.5
12.5
18.2
LAF l LAF 2
Chaetomium nigricolor Penicillium Aspergillus Aspergillus Aspergillus Aspergillus
7.0 151.0 129.5 126.0 36.5 73.5
21.0 180.5 119.5 208.0 66.0 95.0
14.0 165.8 124.5 167.0 51.3 84.3
0 81.5
0 70.8
7.0 118.3
24.0 6.5
0 60.0 56.5
0 0
41.0 0 0
40.3 23.8 0 0
95.4 25.7 42.2
68.4
75.0
71.8
7.9
14.2
11.0
41.4
LAF 3 LAF4 LAF 5 LAF 6
Mean
Mean Mean Mean Mean Mean
for for for for for
Bacillus (LAB l - LAB g) Streptomyces (LAS l - LAS 2 ) Chaetomium (LAF l ) Penicillium (LAF 2) Aspergillus (LAF 3-LAF6 )
For phosphate (P) For Organism (0) Interaction (P x 0) - two P at same 0 Interaction (P x 0) - two 0 at same or different P Interaction (P x 0 x I) - two I at same P and 0 Interaction (P x 0 x I) - two 0 at same P and I Interaction (P x 0 x I) - two P at same 0 and I
B-7653 to B-7658 are CMI code numbers.
82.4
Mean
Mean
Mean for two
61.0 26.7 14.0 Hi5.8 106.8
3.3 7.5 0 70.8 16.0
32.2 17.1 7.0 118.3 61.4
CD at 5%
CD at 1%
25.5 16.7 23.7 28.1 32.0 32.7 35.7
128.2 24.2 34.3 60.5 46.2 47.4 101.2
Phosphate-solubilizing Pnt eut iu lit y of t hr- :\li('l'oorganisluR
21
'fable 4. Organic acids, produced in sucrose-culr-ium phosphate brot h uf'tr-r ten days' incubation by the microorganisms, isolated in sucrose-aluminium phosphate agar plates Organisms
Organic aeids produced
Coded as
Identified as
Identified as
Amount in mg/0.15 g sucrose consumed
LAB l
B acillus subtilis B-7653
Oxalic Succinic
2.775 0.938
LAB 2
Bacillns
Oxalic
UOO
LAB a
Bacillns snbtilis B-7fi54
Oxalic
2.513
LAB4
Bacill us subtilis B-7fi55
2-ketoglueonic Succinic
3.375 4.538
LAB 5
Bacillns
2-ketogluconie Succinic
2.400 3.488
LAB G
BacilluH
Oxalic
1.650
LAB)
Bacillns subtili« B-7(j57
Oxalic
3.300
LABs
Bacillus subtilis B·7658
2-ketogluconic
3.300
LAS l
Streptomyces
Oxalic
1.800
LAS 2
Streptomyces
Oxalic
2.100
LAF l
Chaetomium. nigricolor
LAF2
Pen'icillium
Citric' 2-ketogluconie
5.250 1.500
LA Fa
Aspergillus
Citric 2-ketogluconic
4.388 4.125
LAF4
Asperqillu»
2-ketogluconic
3.150
LAl<\ LAF G
A sperqilius
2-ketogluconic
2.175
Asperqilius
2-ketogluconic
4.200
- indicates organic acid not detected. B-7fi53 to B-7fi58 are CMI code numbers.
Microorganisms produced oxalic, succinic, citric, and 2.ketogluconic acid in sucrose-calcium phosphate broth (Table 4). Although the amount of free organic acid was not correlated with phosphate solubilization, the efficient solubilizers produced 2-ketogluconic acid either singly or in combination with succinic or citric acid. No aliphatic organie acid was produced by Chaeiomium niqricolor in detectable amounts.
Discussion Perusal of the results reveal aluminium phosphate solubilizers to be fewer in population in comparison to Ca3(P0 4)2 solubilizers, and although the organisms were isolated on AIP0 1 , they were more efficient in solubilizing Ca3(PO j )2 ' The indicates that the phosphate solubilizers in the lateritic soil studied were principally Ca3(P() 4)2 solubilizers, They extended their ability to other insoluble phosphate only when its
22
S. BANIK and B. K. DEY
availability became limited. Isolation of the spore formers indicated the prevailing stress condition (ALEXANDER 1977) in the soil studied, especially in terms of texture, pH, organic matter, and nitrogen content (BANIK and DEY 1981). The reason for the morphological variation of some bacilli remained obscure and awaits further investigation. Despite being scant in number, the fungi Penicillium showed the highest solubilizing ability, most probably by virtue of their production of higher amounts of aliphatic organic acids, as suggested by earlier workers (DUFF et al. 1963 ; JOHNsrox 1954). Sustaining the findings by ANAND and JHA (1968), occasional species of Bacillus brought appreciable amounts of soluble phosphorus from insoluble phosphatic sources. The newly found Chaetomium nigricolor was not so efficient in this respect, presumably due to its inability of producing aliphatic organic acids in considerable amounts. Although no generalization in terms of efficiency can be drawn for a genus or species as regards production of a particular organic acid, it was well established by the previous work (BANIK and DEY 1981) and the present data that the organisms, producing 2-ketogluconic acid in combination with succinic or citric, were very efficient in solubilizing insoluble inorganic phosphates. The impediment to AIP0 4 solubilization in broth may be ascribed to the presence of CaC0 3, deliberately added to the broth to maintain the pH, as the first attacking site of the organic acids was supposed to be carbonate instead of phosphate (ANAND and JHA 1968). Nevertheless, the presence of available phosphate in the medium as Ca(H 2P0 4)+ and Ca(HP0 4 ) by the action of free H+, liberated due to partial hydrolysis of AIP0 4 during autoclaving, might have restrained the organisms from utilizing the insoluble phosphate through solubilizing at least in the earlier stage of growth. Inhibitory effect of the consequent Al+3 ion, possibly to a toxic level, to the metabolism of the organisms (ZAJIC 1967) might have played an important role on the variation in morphology of the bacteria. Increase in solubilization of AIP0 4 with the incubation time may be ascribed to the activity of the resistant forms. From the foregoing discussion it becomes clear that tropical soil phosphate solubilizers are essentially Ca3(P04}z solubilizers: they attack other insoluble sources when the earlier substrate is unavailable. However, the exact role of such microorganisms may go beyond expectation in natural soil condition. So, more extensive research in the field will be needed for better understanding of such microorganism - soil relationship and their beneficial role to crop plants. Acknowledgement vVe are grateful to Council of Scientific and Industrial Research, India, for financial help.
References ALEXANDER, M.: Introduction to soil Microbiology. John Wiley & Sons, Inc., New York and London 1977. ANAND, N., and JHA, K. K.: Solubilization of rock phosphate by microorganisms isolated from Bihar soils. J. Gen. Appl. Microbiol. 14 (1968),89-95. BANIK, S., and DEY, B. K.: Phosphate-solubilizing microorganisms of a lateritic soil. 1. Solubilization of inorganic phosphates and production of organic acids by microorganisms, isolated in sucrose calcium phosphate agar plates. Zbl. Bakt. II 136 (1981),478-486. BERGEY'S Manual of Determinative Bacteriology, 8th edn. (BUCHANAN, R. E., and GIBBONS, N. E., eds.), Baltimore 1974. BL:NT, J. S., and ROVIRA, A. D.: Microbiological studies of some subanturr-t.ie soils. J. Soil Sci. 6(1955), 119~128. CHANG, S. C., and JACKSON, M. L.: Fractionation of soil phosphorus. Soil Sci. 84 (1957), 133-144 DUFF, R. E., WEBLEY, D. M., and SCOTT, R. 0.: Solubilization of minerals and related minerals by 2-ketoglueonic acid producing bacteria. Soil Sci. 95 (1963), 105-114.
Phosphate-solubilizing Potentiality of t hr- Microorganisms
2~
GILMAN, J. C.: A manual of soil fungi. 2nd rev. edn. Iowa State College Press 1957. HAYMAN, D. S.: Phosphorus cycling by soil microorganisms and plant roots. Soil Miembiology (WALKER, N., ed.). Butterworths, London 1975, 67 -92. JACKSON, M. L.: Soil chemical analysis. Prentiee-Hall of India Pvt. Ltd., New Delhi 1967. JENSEN, H. L.: Azotobacteriaceae. Bact. Rev. 189 (1930), 195-2.14. JOHNSTON, H. W.: The solubilization of insoluble phosphate. II. A quantitative and comparative study of the action of seleeted aliphatic acids on tricalcium phosphate. N.Z.J.Sei. Teehn. 36 (1954),49-55. MARTIN, J. P.: Use of acid, rosobengal and streptomycin in the plate method for estimating soil fungi. Soil Sci. 69 (1950), 215 - 232. PIKOVSKAIA, R. 1.: Mobilization of phosphates in soil in «onnection with vital aetivity of some microbial species. Mikrobiologiya 17 (1948), 362-370. REID, R. L., and LEDERER, M.: Separation and estimation of sat.urod Cz -C 7 fatty acids by paper partition chromatography. Biochem. J. 50 (1951), 60-67. RlJDRA, P.: Characteristics and genesis of some of the lateritie soils of West Bengal, leading to their elassifieation and land use planning. Ph.D. Thesis, Calcuttu University, Calcutta, India (1975). RUSSELL, E. VV.: Soil condition and plant growth. 10th edn. New York 1973. SPERBER, J. 1.: The incidence of apatite solubilizing organisms in the rhizosphere and soil. Aust. J. Agrie. Res. 9 (1958),778-787. TlJITE, J.: Plant Pathological Methods - Fungi and Bacteria, Burgess Publishing Co., Minneapolis 1959, 239. WAKSMAN, S. A.: The Aetinomyeetes. Ronald Press Co., New York 1967, 280. ZAJIC, J. E.: Microbiol Biogeochemistry, Academic Press. New York and London 1967, 345. Eingegangen am 8. 2. 1982. Authors' addresses: Dr. S. BANIK, University College of Agrieulture, Microbiologieal Laboratory, Calcutta University, Calcutta 700019, and Dr. B. K. DEY, Department of Agricultural Chemistry and Soil Scienee, Faeulty of Agriculture, Bidhan Chandra Krishi Viswa Vidyalaya, Kalyani, Nadia, Pin 741235, West Bengal, India.