INTRODUCTION:

Phosphorus is an essential nutrient for plant growth and development. It is second only to nitrogen in mineral nutrients most commonly influencing growth of crops¹. Phosphorus plays a key role in the balanced nutrition of plants and it is highly insoluble and unavailable to plants². Different bacterial species uses different acids such as gluconic acid, lactic acid, keto gluconic acid, oxalic acid, lactic acid, glycolic acid, acetic acid, succinic acid to solubilize insoluble inorganic phosphate compound (TCP) to soluble organic phosphate¹. The concentration of soluble phosphate in soil is 1.0 ppm or less³. Therefore the application of phosphate fertilizer is essential for enhancing crop growth. Biofertilizer are the formulation of living microorganisms which are able to fix atmospheric nitrogen in the available form for plants (Nitrate form) either by living freely in the soil or associated symbiotically with plants, although nitrogen fixers are present in the soil⁴. Enrichment of soil with effective microbial strains is much beneficial for the crop yields. It has been proved that biofertilizers are cost effective, cheap, and renewable source to supplement the chemical fertilizer⁴.

The genus Pseudomonas, Azospirillum, Bacillus, Rhizobium, Burkholderia, Arthrobacter, Alcaligenes, Serrantia, Enterobacter, Acinetobacter, Flavobacterium, and Erwinia⁵were found to be having phosphate solubilizing potency. Also the Biofertilizers are extremely beneficial in enriching the soil with the microorganism which produce organic nutrient for the soil and help to combat diseases like blue baby syndrome, hypoxia, eutrophication, discolored ground water etc. It is the cheapest source and does not produce any traces of hazardous and poisonous material. Over these advantageous points this study was aimed to isolate strains from various soil sources and to determine their ability to solubilize TCP.

METHODOLOGY:

Collection of Soil Samples:

The soil samples were collected from rhizosphere of different plants at depth of 6-15cm in different area according to the standard procedure⁶ and the sample ID was given.

Isolation of different microbes:

10 gm of soil was taken and serially diluted up to 10^-1-10^-5 dilution. 1ml of aliquots were transferred to Petri plates containing nutrient agar and incubated at 28ºC. After 48h the colony forming units were observed⁵. The colonies which displayed zone were picked up by sterile loop and inoculated in Pikovskaya’s broth and incubated at 28°C for 24 h.

Preliminary screening for phosphate solubilization:

The Pikovskaya’s broth containing phosphate solubilizing microbes were further tested for effective solubilization in Pikovskaya’s agar. The isolates were screened by formation of zone in PVK agar medium incorporated with Tri Calcium Phosphate (TCP). Formation of clear zone around the bacterial growth after seven days of incubation indicates phosphate solubilizing ability⁷.

Solubilization efficiency (%)₌ Diameter of colony + Halozone / Diameter of colony

Microbes exhibiting Phosphate solubilisation were isolated and subjected to characterization by various biochemical and staining techniques.

Preparation of pikovskaya’s agar media for phosphate estimation:

It is the general medium for selection of phosphate solubilizers. In this study Pikovskaya’s (PVK) medium was incorporated with Tri Calcium Phosphate (TCP). The composition’s were Glucose- 10.0gm, Ammonium sulphate- 0.5gm, Sodium chloride- 0.2gm, Magnesium sulphate- 0.1gm, Potassium chloride- 0.2gm, Yeast extract- 0.5gm, Manganese sulphate- Trace, Ferrous sulphate- Trace, Agar- 15.0gm, water- 1000ml. The pH was maintained at 7. The PVK media was prepared in 250ml conical flask using distilled water, sterilized, poured in plates along with, stock suspension of 2.5% TCP and allowed for solidification. Using L-rod the isolated strains were inoculated and incubated at 28ºC for 48h. The formation of zone around the colonies was observed and the diameters were measured.

Preparation of Pikovskaya’s broth for phosphorus estimation:

Sub cultured strains in PVK medium was taken, added 10 ml of Tri Calcium Phosphate (TCP) solution equivalent to 100 mg standard P separately in 90 ml of pikovskaya’s broth. Single colony was inoculated in to 100 ml pikovskaya’s broth medium and incubated at 28ºc in a rotary shaker at 200 rpm. For phosphate solubilization the cultures were harvested every alternative days, centrifuged at 10000 rpm for 15 minutes and the cell free culture filtrates were subjected for phosphate estimation. From the cell free culture filtrate 1ml was used for phosphate estimation by Vandomolybdate blue method⁵.

Study on various parameters with respect to solubilization of tri calcium phosphate in pvk broth containing strains:

Determination of pH:

The pH of the PKV broth was measured by pH meter⁸.

Estimation of soluble phosphate in culture filtrate

From the PVK broth filtrate, 100µl was taken in 50ml of colored volumetric flask, and added with 10 ml of chloromolybdic acid reagent along the sides of the flask, Diluted the contents of the flask to 40 ml. Mixed with 1 ml of chlorostannous acid reagent. After mixing the volume was made to 50 ml as quick as possible. The blue color intensity of the solution was measured at 600 nm against blank (without culture filtrate) and interpreted with the standard graph.

Determination of acid phosphatase enzyme activity:

Pikovskaya’s broth with strain was washed aseptically with distilled water followed by a rinse with filter sterilized modified universal buffer. 1ml of the broth was placed in 30 ml screw cap tubes with 4 ml of sterile buffer solution. 1 ml of filter sterilized 0.115 M disodium p-nitro phenyl phosphate solution was added. The contents were incubated at 30°C in a water bath for 2 hours in the dark. After 2 hours incubation was terminated by adding 5 ml of 0.5 N NaoH. Intensity was measured with UV-VIS spectrophotometer at 410 nm. Acid phosphatase activity was indicated as the amount of p-nitro phenol released in the filtrate from the p-nitro phenyl phosphate per gm of the broth. The p- nitro phenol was calculated with reference to a calibrated graph plotted from the results obtained by standards containing 0, 10, 20, 30, 40, 50 µg of p-nitro phenol. To perform controls, the procedure was followed as described for the assay but made the addition of 1 ml p-nitro phenyl phosphate after the addition of 0.5N NaoH. Phosphatase activity was calculated in ppm/l.

Chemical Analysis:

The PVK broth supernant was taken after centrifugation and estimated for presence of organic acid by performing chemical tests (Biuret, Molisch, Ferric chloride, Seliwanoff’s). The change in colour inferred the presence of organic acids produced by phosphate solubilizing microbes.

Biofertilizer Development:

Carrier Preparation:

Charcoal was weighed and sterilized. The broth was aseptically mixed with carrier manually till it receives 40-50 % moisture and kept for 2-7 days for multiplication of Rhizobium and this process is called curing. Gum arabic and calcium carbonate mixed together to form a slurry. Seeds are soaked in the slurry for 1 day and dried in shade. Pellets were formulated on seeds and sowed in soil, mixed along with Biofertilizer for growth⁴.

Crop growth analysis:

A nursery experiment was undertaken to evaluate the effectiveness of the bacteria in improving the growth and phosphorus levels in paddy seedlings in Tri Calcium Phosphate amended with soil. The seeds of paddy were planted and grown for 3 weeks. Various growth parameters tested in crop growth analysis¹, such as Germination of seed, colour of the crop and Height.

Table 1: SOLUBILIZATION ZONE OF DIFFERENT WILD STRAINS IN PVK AGAR PLATE

S. NO	Sample ID	Diameter of zone for P solubilization(mm) in days
S. NO	Sample ID	1	2	3	4	5	6	7
1	BAR-01	1	1.35	1.77	1.83	2.03	2.24	2.33
2	KRC-01	2.14	2.50	3.02	3.73	4.12	4.96	5.0
3	VEN-01	1.2	1.64	1.89	2.41	2.65	2.89	3.02
4	VED-01	1.44	1.88	2.43	2.62	3.20	3.62	4.11
5	VEH-01	1.02	1.21	1.50	1.80	1.98	2.17	2.31
6	VED-02	2.15	2.45	2.90	3.92	4.09	4.64	4.77
7	KRC-02	2.55	3.17	3.55	3.89	4.38	4.99	5.08

Table 2: DETERMINATION OF pH IN PVK BROTH

S. No	Sample ID	Day1	Day2	Day3	Day4	Day5	Day6	Day7	Day8	Day9
1	Control	7.00	7.00	7.00	7.00	7.00	7.00	7.00	7.00	7.00
2	KRC-01	7.00	5.36	5.01	4.53	4.13	3.91	3.29	3.10	5.88
3	VEN-01	7.00	6.56	5.88	5.46	5.05	4.43	4.12	3.32	6.16
4	VED-01	7.00	6.13	5.99	4.85	4.65	4.20	4.06	3.06	5.36
5	KRC-02	7.00	6.27	5.40	4.44	4.26	3.85	3.26	3.05	5.53
6	VED-02	7.00	5.86	5.01	4.71	4.79	4.56	4.00	3.06	5.40

Table 3: ESTIMATION OF PHOSPHORUS

S. No	Sample ID	Day2 (µg)	Day4 (µg)	Day6 (µg)	Day8 (µg)
1	KRC-01	81.21	98.7	136.09	162.43
2	VEN-01	70.24	89.9	136.09	142.6
3	VED-01	68.04	129.5	140.4	160.23
4	KRC-02	87.8	153.65	160.23	166.82
5	VED-02	76.8	81.21	142.6	153.65

Table 4 : ESTIMATION OF PHOSPHATASE ENZYME ACTIVITY

S. No	Sample ID	Day 2 IU	Day 4 IU	Day 6 IU	Day 8 IU
1	KRC-01	24	27	30	34
2	VEN-01	19	28	30	32
3	VED-01	21	22	29	34
4	KRC-02	22	28	19	40
5	VED-02	16	24	16	27

RESULTS AND DISSCUSION:

Phosphate solubilizing microbes are a low cost solution that enriches the soil giving a thrust to economic development without disturbing ecological balance. So this investigation was framed to determine the efficiency of soil microbes in phosphate solubilization.

The sample ID were given according to the soils collected from the areas as follows BAR = Bangalore Rose Plant soil, KRC = Kerala Coconut tree soil, VEN = Vellore Neem tree soil, VED = Vellore Drum stick tree soil, VEH = Vellore Hibiscus Herb soil. Adjacent numbers were given to the ID according to the colonies displayed in preliminary screening. The colonies displayed by different bacteria after spreading them to the petriplates containing Nutrient agar media shows the zone which reveals the phosphate solubilization efficiency of the same. Those organisms were characterized and they were Pseudomonas fluorescence- KRC 01, Pseudomonas species- VED 01, Bacillus subtilis- VEN 01, Pseudomonas aeruginosa- KRC 02, Bacillus cereus- VED 02 and E.coli- VEH 01. Application of PSMs in the field has been reported to increase crop yield, several like lowering of pH by acid production, ion chelation and exchange reactions⁶. Phosphate solubilizations by strains (Pseudomonas fluorescence-, Pseudomonas species, Bacillus subtilis, Pseudomonas aeruginosa, Bacillus cereus and E.coli) were indicated (Table 1) by hallow formation around the growth of organisms in PVK agar media with 2.5% Tri Calcium Phosphate (TCP). Solubilization index of selected strains of phosphate solubilizing bacteria in PVK agar plates was calculated according to the standard formula⁶. Samples which produced maximum clearing zone (KRC-02, KRC-01, VED-01, VED-02 and VEN-01) in plate assay were further tested for P-Solubilization in Pikovskaya’s (PVK) broth. The principal mechanism for Mineral phosphate solubilization (MPS) is the production of organic acids results in acidification of the microbial cell and its surrounding⁹ Gluconic acid seems to be the most frequent agent of MPS¹⁰. Also keto gluconic acid is another organic acid identified in strains with PS ability¹¹. The pH changes in PVK broth, as the changes in pH determine the production of organic acids in the broth which is the major parameter for the conversion of insoluble phosphate to soluble phosphorus⁶. From our results tabulated (Table 2) the isolated strains shows lowering in pH to 8^th day were, KRC-02 = 3.05, KRC-01= 3.10, VED-01= 3.06, VED-02= 3.06 and VEN-01= 3.32. A fall in pH accompanied phosphate solubilization, due to production of organic acids was observed up to 8 days, but there is a sudden increase in pH after 9^th day. This may be due to utilization of organic acid produced during phosphate solubilization by these strains. The release of soluble phosphorus in the culture filtrate of PVK broth by selected phosphate solubilizing micro organisms isolated from different soil rhizospheres shows change in releasing of phosphorus on 4^th day. From Table 3 (KRC-01= 98.7µg, KRC-02= 153.65µg VED-01= 129.5µg, VED-02= 81.21 µg and VEN-01= 129.5 µg) a gradual increase in phosphorus content was observed on 8^th day KRC-01= 162.43 µg, KRC-02= 166.82µg, VED-01= 160.23µg, VED-02= 153.65 µg and VEN-01= 142.06 µg. Among the 5 isolates only 3 exhibited significant releases in phosphorus level (KRC-02, KRC-01 and VED-01). Solubilization kinetic studies confirmed the release of soluble phosphorus by KRC-02= Pseudomonas aeruginosa, KRC-01= Pseudomonas fluorescence, VED-01= Pseudomonas species, VEN-01= Bacillus subtilis and VED-02= Bacillus cereus. In PVK broth, blended with TCP these solubilizers exhibits increased in release of soluble phosphorus right from fourth to eighth day. So by the investigation we can infer that the phosphorus released by these strains was successfully correlated with the standard limits. The major enzyme which involves in the reaction of organic acid conversion is phosphatase¹². On determining the activity of enzyme will be a sound proof in solubilizing efficiency of microbes. From Table 4 it is understood that, the release of soluble phosphorus in the culture filtrate of PVK broth by selected phosphate solubilizing micro organisms isolated from different soil rhizospheres shows change in releasing of phosphorus by enzyme. On 4^th day KRC-01= 27 IU, KRC-02= 28 IU, VED-01= 22 IU, VED-02= 24 IU and VEN-01= 28 IU. The results showed gradual increase on 8^th day were KRC-01= 34 IU, KRC-02= 40 IU, VED-01= 34 IU, VED-02= 27 IU and VEN-01= 32 IU. Among the five isolates only three exhibits better results than remaining two. Acid phosphatse enzyme activity was observed by all strains. The observation of enzyme activity was predominant on 8^th day in PVK broth containing TCP (2.5%) and given in international units. Solubilization kinetic studies confirmed the phosphatase enzyme activity plays a major role in realizing the organic acid by KRC-02= Pseudomonas aeruginosa, KRC-01= Pseudomonas fluorescence, VED-01= Pseudomonas species, VEN-01= Bacillus subtilis and VED-02= Bacillus cereus. In PVK broth, blended with TCP these solubilizers exhibits the increased enzyme activity right from fourth to eighth day. So by the results we can infer that the phosphatase enzyme catalyst converts the microbial strain successfully to perform the solubilization of phosphorus by releasing acids. From the literature studies it was known that the Phosphate solubilizing microbes use glucose as carbon source to produce their own specific organic acid for the conversion of phosphorus¹³. So the experiment was designed according to that for the detection of respective acids. From our experimental reports the organic acids were determined and the organisms were confirmed vice versa. The nursery experiment was under taken to evaluate the effectiveness of these bacteria in improving the growth and phosphate level of paddy seedling in TCP amended soil. The results for crop growth analysis was shown by seed germination on first week for KRC-01 = 5 days, VED-01 = 6 days, KRC-02 = 5 days, VEN-01 = 6 days, VED-02 =6 days, chemical fertilizer (CF) =10 days, soil =8 days and control =11 days, followed by color change for KRC-01, KRC -02, VED-01, VEN-01 and VED-02 shows dark green color comparatively tested (soil, chemical fertilizer, control) showed pale green color. The crop height measured for first week were, KRC-01 = 4cm, KRC-02 = 4.5cm, VED-01 = 4cm, VEN-01 = 2cm and VED-02= 3cm others showed comparatively less growth. The third week growth analysis for color change were observed as dark green for KRC-01, KRC-02, VED-01, VED-02, VEN-01 and Chemical fertilizer(CF) the other two treatment (soil and control) shows pale green color. And the crop height showed gradual increase for KRC-01 = 19.6cm, KRC-02 = 21 cm, VED-01 = 20cm, VEN-01= 18cm, VED-02 = 19cm, Soil= 16cm, Chemical fertilizer= 17cm and Control = 14cm. Comparatively Biofertilizer showed predominant results than other treatments.

CONCLUSION:

From our experimental studies we can conclude that the isolated strains exhibits high degree of tri calcium phosphate solubilisation and can be utilized in bio fertilizer development.

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Received on 25.06.2010 Modified on 06.07.2010

Asian J. Research Chem. 3(4): Oct. - Dec. 2010; Page 1054-1057