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www.sciencejournal.in ISOLATION AND CHARACTERIZATION OF A NOVEL MULTIFUNCTIONAL SULPHUR OXIDIZING BACTERIUM (SOB) AND ITS USE AS BIOFERTILIZER S. Priyanka*, M. Sivaji and R. Sridar Department of Plant Biotechnology, Centre for Plant Molecular Biology and Biotechnology, Tamil Nadu Agricultuural University (TNAU), Coimbatore-641003. *(Corresponding author: [email protected]) ABSTRACT The Sulphur oxidising bacteria (SOB) have been isolated from 8 different samples viz., match factory, sewage, paper effluent, treated tannery effluent, untreated tannery effluent, rhizosphere soil, acidic soil and treated beverage waste. The isolates were checked for their ability to oxidise sulphur and solubilise insoluble phosphate, nitrogen fixation and antagonistic activity against plant pathogenic fungi like Rhizoctonia solani and Macrophomina phaseolina and Fusarium solani. The obtained isolates were characterized using biochemical tests, temperature and pH tolerance tests and screened by PCR using specific primers (soxB). KEY WORDS: antagonism, Nitrogen fixation, phosphate solubilisation, Sulphur oxidization. INTRODUCTION Sulphur is now considered as the fourth essential nutrient after N, P and K, especially in agriculture crop production. It is a constituent of the essential amino acids like Cysteine and Methionine and plays an important role in the formation of proteins, vitamins and enzymes. Sulfur is an acidifier which reduces the pH of calcareous and alkaline soils and facilitates the absorption of some nutrients. Sulfur also improves the condition of sodium soil and controls some plant pathogens. (Kertesz et al., 2004). The sulfur bacteria comprise a heterogeneous group of organisms which share the ability to oxidize reduced or partially oxidized inorganic sulfur compounds. Thiobacilli play an important role in sulphur oxidation in soil which improves soil fertility. It results in the formation of sulphate, which can be used by the plants, while the acidity produced by oxidation helps to solubilize plant nutrients and improves alkali soils (Hitsuda et al., 2005). The sulphur bacteria can grow in a wide pH range of 1.0 -10.5. The sulphur oxidizing microorganisms are primarily the Gram negative bacteria, classified as species of Acidothiobacillus, Thiomicrospira and Thiosphaera, but heterotrophs, such as some species of Paracoccus, Xanthobacter, Alcaligens and Pseudomonas can also exhibit chemolithotrophic growth on inorganic sulphur compounds (Stamford et al., 2003). Thiobacillus thiooxidans, T. ferrooxidans, T. thioparus and T. denitrificans are obligate chemoautotrophs while T. novellus is considered a facultative chemoautotroph. The main objective of this study is to isolate sulpur oxidising bacteria and to select the efficient isolates from them for higher sulphur oxidation, phosphate solubilization and antagonistic properties to promote the growth of plants. MATERIALS AND METHODS Sample Collection Autotrophic Sulphur oxidising bacterial isolates were obtained from 8 different samples viz., match factory (TSM), sewage (TSS), paper effluent (TSP), treated tannery effluent (TST), untreated tannery effluent (TSU), rhizosphere soil (TSD), acidic soil (TSV), treated beverage waste (TSB). Isolation and selection of SOB The procedure described by Vidyalakshmi and Sridar (2007) was adopted for the isolation of sulphur oxidising bacteria. Starkey broth and Sodium thiosulphate broth without glucose was the two different selective broth employed for isolation.The Sulphur oxidising isolates thus obtained were purified and used for characterization and for further studies. The pure cultures were inoculated in the growth media with initial pH adjusted to 8.0 and incubated at room temperature for 25 days. The final pH of the growth media was measured using a pH meter. The isolates were screened for their efficacy to reduce the pH from 8.0 to 4.5. The selected isolates were further studied for their morphological and biochemical characterization.

Volume- 1 Issue-1 (2014)

ISSN: 2348 – 604X (Print); 2348 – 6058 (Online)

© 2014 DAMA International. All rights reserved

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www.sciencejournal.in Morphological and Biochemical Characterization The selected isolates were plated in sodium thiosulphate agar medium with pH 8.0 to study colony morphology. The isolates were subjected for Gram staining and Motility test. Also biochemical tests like catalase, MR-VP, starch hydrolysis, citrate utilization tests, H2S production tests, urease test, nitrate reduction test, triple sugar iron agar test (TSI) were performed and the results were recorded. Quantitative estimation of Sulphate In order to evaluate the utililsation of sulphur by the isolates quantitatively, turbidimetry method was followed. A quantity of 5ml of 24 hours old culture was taken and to which 0.15% CaCl 2 (25ml) was added and kept on a shaker for 30 minutes. From this 5 ml of the extract was taken in a 25ml volumetric flask to which 10 ml sodium acatate buffer, 1 ml gum acacia and 1g BaCl2 was added. Absorbance was read at 420 nm in a spectrophotometer and the sulphate content was calculated using standard curve. Estimation of Soluble Phosphorous The purified isolates were tested for phosphate solubilisation in the Sperber’s hydroxy apatite medium (HAP). The cultures were streaked on HAP plates and incubated at 37oC for 2-3 days for assesing the ‘P’ solubilizing ability of the isolates. The zone of phosphate hydrolysis was measured and tabulated. Quantitative assay of phosphorous estimation was carried out using the method described by (Olsen et al., 1954). Nitrogen Fixation The N2 fixing ability of the isolates was identified by growing the cultures in a N free BMGM broth (pH 5.7) using Bromothymol blue as indicator with malate as a carbon source. The sterilized BMGM broth was inoculated with the test isolates and incubated at 370C for 10 days. The alterations in pH were recorded at an interval of every 2 days and results were tabulated (Govindarajan et al., 2007). Temperature and pH Tolerance Test Temperature and pH tolerance tests were performed for 8 isolates at different temperatures like 4 0 C, 280 C, 370 C, 450 C and growth was observed. Similarly they were grown at different pH ranginig from 2-8 and growth was monitored. Antagonism and IAA production The SOB isolates were tested for their ability to control the growth of fungal pathogen like Rhizoctonia solani and Macrophomina phaeseolina. The antagonistic activity of the isolates was determined by dual culture method using sodium thiosulphte agar and potato dextrose agar medium in the ratio 1:1 (Rangeswaran and Prasad, 2000). Genomic DNA isolation and Molecular characterization of isolates: Total genomic DNA was isolated from SOB isolates. Genomic DNA isolation was carried out from the isolates by using the phenol/chloroform/ isoamyl alcohol method (Lazo et al., 1987). The total genomic DNA isolated from SOB isolates was amplified by PCR. Polymerase Chain Reaction was performed using the Eppendorf Master Cycler, Gradient (Eppendorf, Germany). PCR amplification was carried out using 16S rRNA universal primer (Morabbi Heravi et al., 2008). Screened isolates were further characterised by amplification with soxB primers. RESULTS AND DISCUSSION Growth in Selective Media The obtained isolates were selected based on the pH reduction test. Among the 8 isolates, 5 isolates viz., TSM, TST, TSS, TSU, TSV caused a pH reduction upto 4.38, 4.49, 4.75, 4.67, 4.94 respectively in Starkey broth (control pH 8.0). Most of the isolates prefer elemental sulphur as sulphur source. The isolates were facultative autotrophic in nature. Isolation of Autotrophic sulphur oxidizers from different ecological niches was reported by Sridar et al. (2013).




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www.sciencejournal.in Table 1. pH Reduction Test in Selective Media S.No

Isolates

1 2 3 4 5 6 7 8 9

TSM TST TSS TSU TSV TSD TSB TSP CONTROL

Starkey Broth (Initial pH 8) 4.38 4.49 4.75 4.67 4.94 5.12 6.95 5.36 8.0

Sodium Thiosulphate Broth (Initial pH 8) 5.53 4.13 6.05 6.90 5.17 6.10 6.0 6.5 8.0

Plate 1. pH reduction by SOB isolates in Starkey media Quantitative Estimation of Sulphur and Phosphate The quantitative estimation of sulphur was calculated using the standard curve.The isolates viz., TSM and TST showed high utilisation of sulphur (Table. 2). Anandham et al. (2005) reported the highest sulfate production by chemolithoautotrophic isolate LCH (43.21 mg 100 mL-1 of broth). Sulfate production from elemental Sulphur in growth media ranged from 31.7 mg 100 mL-1 to 118.0 mg 100 mL-1 by Sulphur oxidizing bacteria (Starkey, 1934). 80 60 40

SULPHATE… PHOSPHATE(mg/1…

20 0 Graph 1: Quantitative estimation of sulphate and phosphate by the SOB isolates Phosphate solubilisation Zone of ‘P’ hydrolysis formed by the isolates in HAP plates was measured following incubation of 3-5 days. The isolate TST formed better halozone which measured 1 cm followed by isolates TSM (0.5cm) and TSU (0.5cm).




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Plate 2: phosphate solubilisation by SOB isolates in HAP medium Nitrogen fixation Nitrogen fixing ability of the test isolates was assessed using BMGM broth (pH 5.7). Among the 8 isolates, 4 isolates viz., TSM, TST, TSS and TSU showed higher nitrogen fixing ability in BMGM broth (Table 2).

C

TSM

TST

TSS

TSU

Plate 3. Nitrogen fixing activity of SOB isolates in BMGM broth Table 2: Isolates Showing Nitrogen Fixing ability in BMGM Broth S.No Isolates 3rd Day 6th Day 9th Day 12th Day 1 2 3 4 5 6 7 8 9

TSM TST TSS TSU TSV TSD TSB TSP CONTROL


6.2 6.3 6.0 6.0 5.7 6.02 5.9 5.7 5.7

7.4 7.0 6.6 6.3 5.7 6.3 5.9 5.7 5.7

7.9 7.65 6.8 6.7 6.4 6.4 6.2 6.0 5.7


8.03 8.0 7.3 7.0 6.8 6.5 6.3 6.2 5.7

15th Day 8.96 8.88 7.5 7.7 6.8 6.5 6.7 6.2 5.7

20th Day 9.02 9.0 7.58 8.0 6.0 6.8 6.7 7.2 5.7


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www.sciencejournal.in Biocontrol Activity against Fungal Pathogens All 8 isolates were tested for antagonistic activity against fungal pathogens Rhizoctonia solani and Macrophomina phaeseolina. The isolates TST inhibited R.solani and zone of inhibition was found to be 0.45cm. The isolate TSM showed antagonistic activity against Macrophomina phaeseolina with an inhibition zone of 0.32cm and no inhibition was found with Fusarium solani.

Plate 4. Biocontrol activity of SOB isolates showing zone of inhibition against R.solani

Table 3. Morphological and Biochemical characterisation of isolates S.No 1 2

Tests Gram reaction Colony morphology

TSM Rods

TST Rods

TSS Rods

TSU Rods

TSV + Rods

TSD Rods

TSB + Rods

TSP + cocci

3 4 5 6 7 8

Catalase MR VP Starch hydrolysis Citrate test H2S production

+ + + + -

+ + + + -

+ + -

+ + +

+ + -

+ -

+ + + + -

+ + + + -

9 10 11

Urease Nitrate reduction TSI

+ Alkaline slant acid butt

+ Alkaline slant alkaline butt

Alkaline slant acid butt



Growth at 40 C Growth at 280 C Growth at 370 C

+ ++ +++

+ ++ +++

+ ++ +++

+ ++ +++

+ Alkaline slant alkaline butt ++ +++


12 13 14

Alkaline slant acid butt ++ +++

+ + +++

++ +++

15

Growth at 450 C

++

++

++

-

++

++

-

-

‘+’= positive result, ‘-’= negative result pH and Temperature Tolerance Test The results of this study revealed that all 8 isolates were able to grow in a wide range of temperature between 4 0 C 450 C, but the optimum being 280 C - 370 C as indicated in Table 4. Also all the 8 isolates showed fastidious growth in a wide range of pH 4.5-8. Biochemical Tests The result obtained from biochemical tests like catalase, MR-VP, starch hydrolysis, citrate utilization tests, H2S production tests, urease test, nitrate reduction test, triple sugar iron agar test (TSI) are furnished in Table 4.




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www.sciencejournal.in 16s rRNA and soxB gene amplification of SOB isolates The selective SOB isolates were subjected to PCR using specific primers like 16S rRNA and soxB primers.When the genomic DNA of selected SOB isolates (TSM, TST and TSU) were amplified by PCR using soxB primers, a major amplification band of 1400bp was observed and 16sr RNA yielded 1200bp band which was notified by running the PCR product on 1.2% agarase gel.

M

1

2

3

Lane M – Marker; 1kb DNA ladder

2000 bp

1000 bp Lane 1 – TST isolate Lane 2 – TSM isolate Lane 3 – TSU isolate Plate 5. Gel electrophoresis of amplified soxB gene of SOB isolates through PCR CONCLUSION Among the 8 isolates, those obtained from match factory (TSM) and treated tannery effluent (TST) was capable of sulphur oxidation, phosphate solubilization, N2 fixation and biocontrol activity. The isolates were able to grow at wide range of pH 2-8 and temperatures between 40 C to 450 C. The present study revealed that the isolates TST and TSM have the potential for developing into a farmer friendly bioinoculant for enhancing the Sulphur and Phosphorus nutrition, growth promotion and for disease management in several crops after testing under field conditions (Anandham et al., 2007). ACKNOWLEDGMENT The authors are grateful to DBT, Govt. of India for providing financial support to carry out this work. REFERENCES Anandham R., Sridar R., Nalayani P., Madhaiyan M., Gandhi P.I., Choi K. and Sa T. (2005). Isolation of sulphur oxidising bacteria from different ecological niches. Korean J. Soil Sci.Fert. 38. Anandham R., Sridar R., Nalayani P., Poonguzhali S., Madhaiyan M. and Tongmin Sa. (2007). Potential for plant growth promotion in groundnut (Arachis hypogaea L.) cv. ALR-2 by co-inoculation of sulfur oxidising bacteria and Rhizobium. J. Microbiol. Res. 162: 139-153. Govindarajan M., Kwon S.W. and Weon H.Y. (2007). Isolation, molecular characterization and growthpromoting activities of endophytic sugarcane diazotroph Klebsiella sp. GR9. World J. Microbiol. Biotechnol. 23: 997-1006. Hitsuda K., Yamada M. and Klepker D. (2005). Sulfur requirement of eight crops at early stages of growth . J. Agron. 97: 155-159. Kertesz M. A. and Mirleau P. (2004). The role of Soil Microbes in Plant Sulphur Nutrition. J. Exp. Botany. 44: 1939-1945. Lazo G.R., Roffey R. and Gabriel D.W. (1987). Conservation of plasmid DNA sequences and pathovar identification of strains of Xanthomonas campestris. J. Phytopathol. 77: 448-453. Morabbi Heravi K., Eftekhar F., Yakhchali B. and Tabandeh F. (2008). Isolation and identification of a lipase producing Bacillus sp from soil. Pak. J. Biol. Sci. 11(5):740-745.




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www.sciencejournal.in Olsen S.R., Cole C.V., Watanable F.S. and Dean L.A. (1954). Estimation of soil phosphorous in soil by extraction with sodium bicarbonate. USDA Circular no. 939. Rangeshwaran R. and Prasad R.D. (2000). Biological control of Sclerotium rot of sunflower. Indian Phytopathol. 53: 444-449. Sridar R., Raveendran M., Sivaji M. and Gayathri R. (2013). Isolation of Autotrophic sulphur oxidizers from different ecological niches. J. Soil Biol. Ecol. 33: 66-76. Stamford N.P., Santos A.M., Moura M.F., Santos C.E.R.S. and Frietas A.D. (2003). Biofertilizers with natural Phosphate Sulphur and Acidothiobacillus in a soil with low available-P. Sci. Agric. 607: 63-73. Vidyalakshmi. R. and Sridar. R. (2007). Isolation and characterization of sulphur oxidising bacteria. J. Cult. Collections. 5: 73-77. Vidhyasri S. and Sridar R. (2011). Evaluation of co inoculation of Sulphur oxidizing bacteria and Rhizobium for enhancing productivity of groundnut. IJTA. 29: 35-40. Vimala P. and Sridar R. (2009). Studies on the use of Sulphur oxidising Bacterial inoculant in Groundnut. J. Soil. Biol. Ecol. 29: 30-41.




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