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Delineation of Sulphur Bacteria Isolated from Rice Rhizosphere Soil

K
K. Sowmya1,*
S
S.L. Sivapriya2
1Department of Agricultural Microbiology, School of Agricultural Sciences, Bharath Institute of Higher Education and Research, Tambaram, Chennai-600 001, Tamil Nadu, India.
2Janani Public School, Bangalore-560 001, Karnataka, India.

ABSTRACT

Background: Crop yields are greatly reduced by sulfur-deficient conditions, as sulfur is an essential nutrient for plant growth. So, sulfur fertilizers are generally added to soils to alleviate this deficiency, usually in a reduced form, such as elemental sulfur. Despite this, microorganisms are required to oxidize reduced sulfur fertilizers into sulfate before they can be absorbed by plants. Recently, sulfur deficiency was reported widely in rice. Till date there are no suitable bio-inoculants for rice. Hence, the present study was aimed to develop a sulfur bio-inoculant for rice.

Methods: In this present investigation, totally 9 chemolithoautotropic sulfur oxidizing bacteria (3 from aerobic, 4 from wet land and 2 from SRI systems) were isolated from various rice ecosystems. Chemolithoautotrophic strain S1-3 isolated from SRI system and strain W3-1 isolated from wet land were found to efficient based on pH reduction and titrable acidity. Totally five facultative chemo lithoautotrophic sulfur oxidizing bacteria (two from wet land and 3 aerobic samples) were recovered from different rice ecosystems.

Result: Facultative chemolithoautotrophic strain S1Y1-bLL and S2Y2-b significantly reduced the medium pH (4.3) and consumed the maximum alkali 1.7 and 1.8 mL and found to be efficient. All the isolated facultative chemolithoautotrophic sulfur were autotrophic and chemoheterotrophic in nature. Molecular characterization study revealed that strains S1Y2 - aS, S1Y1 - Bll, S2Y2 - b, S2SA - Sa and S2Y2 - an exhibited 99.5, 99.7, 99.5, 99.6 and 99.9% sequence similarities to Pseudomonas beteli, Advenella kasmirensis, Pseudomonas beteli, Providencia stuartii and Bacillus tequilensis, respectively. Further, developed lignite and compost based and clay pellet formulation for facultative chemolithoautotrophic sulfur oxidizing bacteria. Yet, plant inoculations studies should be carried out for isolated sulfur oxidizing bacteria from this study before recommended to the farmers.

INTRODUCTION

In addition to nitrogen, phosphorus and potassium, sulfur (S) is becoming increasingly recognized as a major plant nutrient. A compound containing reduced sulfur is produced from the amino acids cysteine and methionine, which act as precursors (Narayan et al., 2023). In addition to holding structural and functional roles within macrobiomolecules, sulfur modulates many physiological processes and makes plants more tolerant to abiotic stress (Haneklaus et al., 2003). Plants and animals require sulfur to survive. A sulfur deficiency in the field was first reported in 1933, despite plants’ need for sulfur in comparable amounts to phosphorus. The first report of sulfur deficiency in wetland rice was made in 1938. Dry-land crops as well as wetland rice have been found to be affected by sulfur deficiency over the last 10 years. The tropics are generally characterized by sulfur deficiency in Andosols, Vertisols, Alfisols, Ultisols and Oxisols. Several Asian countries, including Bangladesh, Burma, India, Indonesia, Japan, Philippines and Sri Lanka, have reported sulfur deficiency of wetland rice (Roy et al., 2017). Twenty-three crops have been reported to respond to sulfur in forty tropical countries (Jenni, 2020). A decrease in organic manures, intensive cropping and decreased atmospheric deposition have contributed to this increase in sulfur deficiencies (Dawar et al., 2023). The use of sulfur fertilizers is invariably used to alleviate sulfur deficiencies in soils, usually in reduced forms, such as elemental sulfur. Use of S oxidizers enhances the rate of natural oxidation of S and speed up the production of sulfates and makes them available to plants at their critical stages, consequently resulting in increased plant yield (Anandham et al., 2008a, b). Micororganisms contribute the biogeochemical cycling of sulfur (Friedrich et al., 2005). Various microorganisms (prokaryotes, green sulfur bacteria) have been described which utilize sulfur compounds obligately or facultatively as electron donors and oxidize them to sulfate. Sulfur oxidizing bacteria such as Bosea thiooxidans, Paracoccus thiocyanatus, Pseudamino- bacter salicylatoxidans, Paracoccus pantotrophus, Paracoccus bengalensis, Tetrathiobacter kashmirensis and Mesorhizobium thiogangeticum were isolated from several ecological niches (Sowmya and Sivapriya 2026).

MATERIALS AND METHODS

Rhizosphere sample collection
 
Rhizosphere soil samples were collected from rice cultivated land at various ecosystems (Wetland, SRI and aerobic) (Table 1). Three plants at flowering stages were selected and removed from the soil, placed in a polythene bag, immediately transported to the laboratory and processed. The plants were shaken gently to separate the soil that was not tightly adhering to the roots. The rhizosphere soil (soil attached to the roots after gentle shaking) and fine roots (approximately 1 cm length) were collected from the plants and used for enrichment isolation (Anandham et al., 2008).

Table 1: Plant source for isolation of sulfur oxidizing bacteria.


 
Enrichment Isolation
 
Enrichment isolation of chemolithoautotrophic sulfur-oxidising bacteria
 
For isolation of chemolithoautotrophic sulfur oxidizing bacteria, three 1 g replicates of each rhizosphere soil samples were added into 10 mL of liquid mineral salts thiosulfate (MST) medium containing (g L-1) NH4CI, 1.0; K2HPO4, 4.0; KH2PO4, 1.5; MgSO4.7H20, 0.5; Na2S2O3.5H20, 5.0; yeast extract, 0.05; bromocresol purple, 0.002; trace element solution 5 ml; pH 7.5). The medium was then incubated at 30oC in the dark to avoid growth of phototrophic bacteria on a rotary shaker (120 rpm) until the color of the bromocresol purple changed to yellow.
 
Enrichment isolation of facultative chemolithoautotrophic sulfur-oxidizing bacteria
 
For isolation of chemolithoautotrophic sulfur-oxidizing bacteria, three I g replicates of each rhizosphere soil sample were added into 10 mL of liquid mineral salts thiosulfate yeast extract (MSTYE) medium containing (g L-l) NH4CI, 1.0; K2HPO4, 4.0; KH2PO4, 1.5; MgSO4.7H20, 0.5; Na2S2O3.5H20, 5.0; yeast extract, 5.0; bromocresol purple, 0.002; trace element solution 5 ml; pH 7.5). The medium was then incubated at 30oC in the dark to avoid growth of phototrophic bacteria on a rotary shaker (120 rpm) until the colour of the bromocresol purple changed to yellow.
 
Purification of sulfur-oxidising bacteria
 
For isolation of pure cultures, streak plate method is followed a Loop full of culture is streaked on the surface of MST/MSTYE agar plates (Fig 6). The colonies that developed yellow halo against purple background, indicative of the production of sulfuric acid resulting from the oxidation of thiosulfate were picked and streaked on solid MST medium until uniform colony morphology was observed. Colonies were transferred 3 times to be considered as pure, in addition, the purity of the strains was checked microscopically. The pure bacterial strains were maintained on MST agar plates, sub cultured every week and subjected to further studies.

Screening of efficient sulfur-oxidizing bacteria
 
pH reduction test
 
Isolated chemolithoautotrophic and facultative chemolitho-autotrophic sulfur-oxidising bacteria were inoculated in MST and MSTYE broth and incubated for 5 days at 30oC. The End of sulfur oxidation is the production of sulfuric acid; hence, the pH of the medium would be reduced. The reduction in pH was recorded using the pH meter (Medox, MX-1292-01, India) at 25oC.
 
Titrable acidity
 
One mL of stationary phase cultures of chemolitho-autotrophic and facultative chemolithoautotrophic sulfur-oxidising bacteria cultivated in liquid MST and MSTYE were collected in a beaker and drops of 0.1% phenolphthalein were added. At this moment, it is colourless. It was titrated against 0.1N NaOH until the appearance of pink colour, which indicates the end of the titration. Titrable acidity indicates the indirect production of acid.
 
Morphological characterization
 
Phenotypic characters such as colony colour, colony diameter, cell shape and Gram staining were performed as per the standard procedure (Smibert and Krieg, 1994).
 
Nutritional characterization of facultative chemoltiho-autotrophic sulfur oxidizing bacteria
 
Facultative cheomolithoautotrophic sulfur oxidizing bacteria were streaked on MSTA (Mineral salts thiosulfate agar), NA (Nutrient agar), TSA (tryptic soy agar) and LBA (Luria Bertani agar) and incubated at 30oC for 5 d then growth was checked.
 
Molecular characterization and phylogenetic analysis
 
The purified bacteria were grown in MST broth and harvested in the log to initial stationary phase. The genomic DNA was extracted using standard methods described (Wright et al., 2017). The genomic DNA was amplified using 16S rRNA universal primer (27f: 5' -AGA GTT TGA TCC TGG CTC AG-3' and 1492r: 5' GGT TAC CTT GTT ACG ACT T-3' ) in a thermocycler (Veriti™ 96-Well Fast Thermal Cycler). The authentication potential of SOB was verified by 16S rRNA amplification followed by sequencing for identification. The obtained sequences were performed with a similarity search using BLAST. A phylogenetic tree was constructed with existing 16S rRNA sequences of SOB from different eubacteria obtained from NCBI GenBank database.
 
Development of formulation
 
Development of powdered formulation
 
Facultative chemolithoautotrophic sulfur oxidizing bacteria grown in nutrient broth were used for formulation development. One kg of powdered, neutralized and/or sterilized lignite and compost were mixed with stationary phase culture (1 x 109 cfu mL-1) under aseptic condition, allowed for 2d and packed.
 
Development of powdered formulation
 
One Kg of clay soil was powdered and sterilized and cooled. Then clay soil was mixed with 400 mL of stationary phase Facultative chemolithoautotrophic sulfur oxidizing bacterial culture (1 x 109 cfu mL-1) and pelletized with hand pelletizer and air dried further cut into approximately 1 cm size and stored.

RESULTS AND DISCUSSION

Isolation of chemolithoautotrophic sulfur-oxidising bacteria
 
In total, 27 rice rhizosphere soil samples were collected from various rice ecosystems, such as aerobic, wetland and SRI rice, of which 25 samples contained the chemolitho- autotrophic sulfur-oxidising bacteria. It was inferred that the change of colour from purple to yellow, which indicated the end of sulfur oxidation, is the production of sulfuric acid, ultimately reducing the pH of the medium, in turn changing the medium colour from purple to yellow (Table 2). Above 25 positive samples, the isolates that showed good growth (3 from aerobic, 4 from wetland and 2 from SRI systems) were taken for purification (Table 3).

Table 2: Isolation of chemolithoautotropic sulfur oxidizing bacteria from different ecosystems.



​

Table 3: Selection of chemolithoautotrophic sulfur oxidizing bacteria for purification.


 
Screening of chemolithoautotrophic sulfur-oxidising bacteria
 
Efficient chemolithoautotrophic sulfur-oxidising bacteria were screened based on pH reduction and Titrable acidity, indirectly indicating sulfur oxidation. Among isolates, strain S1-3 isolated from the SRI system showed the maximum reduction of pH (5.4), followed by the wetland strain W3-1 (5.6). Strains W3-1 and S1-3 consumed the maximum alkali 2 and 1.2 mL, respectively (Table 4).

​

Table 4: Screening of chemolithoautotrophic sulfur oxidizing bacteria based on pH reduction and titrable acidity.


 
Isolation of facultative chemolithoautotrophic sulfur-oxidising bacteria
 
In total, 27 rice rhizosphere soil samples were collected from various rice ecosystems, such as aerobic, wetland and SRI rice. Of which 5 samples (two wetlands and 3 aerobic samples) contained the efficient facultative chemolithoautotrophic sulfur-oxidising bacteria (Table 5). Above 5 positive samples, 5 facultative chemolithoautotrophic sulfur-oxidising bacteria were recovered (Table 5; Fig 1).

​

Table 5: Isolation of facultative chemolithoautotrophic sulfur oxidizing bacteria.



Fig 1: Purification of facultative chemolithoautotophic Sulfur oxidizing bacteria.


 
Screening of facultative chemolithoautotrophic sulfur-oxidising bacteria
 
Efficient facultative chemolithoautotrophic sulfur-oxidising bacteria were screened based on pH reduction and titrable acidity, indirectly indicating sulfur oxidation. Strain S1Y1-bLL and S2Y2-b significantly reduced the medium pH (4.3) and consumed the maximum alkali 1.7 and 1.8 mL (Table 6).

​

Table 6: Screening of facultative chemolithoautotrophic sulfur oxidizing bacteria based on pH reduction and titrable acidity.


 
Morphological characterisation of facultative chemolitho-autotrophic sulfur-oxidising bacteria
 
Among the screened facultative chemolithoautotrophic sulfur-oxidising bacteria, all were Gram-negative rods except strain S2Y2-a, which was Gram-positive (Table 7).

​

Table 7: Morphological characterization of facultative chemo lithoautotrophic sulfur oxidizing bacteria.


 
Nutritional characterisation of facultative chemolitho-autotrophic sulfur- oxidising bacteria
 
All the isolated facultative chemolithoautotrophic sulfur-oxidising bacteria strains were able to grow on autotrophic medium (MSTA) as well as on NA, TSA and LBA. This indicated that all the facultative chemolithoautotrophic sulfur oxidizing bacteria isolated from this study were autotrophic as well as chemoheterotrophic in nature (Table 8; Fig 2).

​

Table 8: Nutritional characterization of facultative chemo lithoautotrophic sulfur oxidizing bacteria.



Fig 2: Growth of facultative chemolithoautotrophic sulphur oxidizing bacteria in NA medium.


 
Molecular characterization of facultative chemolitho-autotrophic sulfur oxidizing bacteria
 
Molecular characterization of sulfur oxidizing bacteria was performed through 16S rDNA sequencing. Sequence results revealed that strains SlY2- as, S1Y1-bLL, S2Y2-b. S2SASa and S2Y2- a cxhibited 99.5, 99.7, 99.5, 99.6 and 99.9% sequence similarties to Pseudomonas beteli, Advenella kashmirensis, Pseudomonas beteli Providencia stuartii and Bacillus tequilensis, respectively. In phylognelic analyses revealed that clustering patern were similar in both neighbor-joining and maximum likely hood trees. Irrespective of phylogenetic methods used, strain S1Y 1- bLL formed cluster with Advenella kashmirensis which was supported by bootstrap value of 100, 92 and 9 in neighbor -joining (NI), maxmimum-likely hood (ML) and maximum- parsimony (MP) trees. Similarly, strains S1Y2- aS and S2Y2- b clustered with Pseudomonas beteli. Strains S2SA -Sa and S2Y2 formed separate clade with Providencia stuartiiand Bacillus tequilensis, respectively (Table 9; Fig 3, 4, 5).

​

Table 9: Molecular characterization of facultative chemolithoautotrophic sulfur oxidizing bacteria isolated from different ecosystems.



​

Fig 3: Neighbour-joining phylogenetic tree based on complete 16S rRNA sequences.



​

Fig 4: Maximum likely hood phylogenetic tree based on complete 16S rRNA sequences.



​

Fig 5: Maximum parsimony phylogenetic tree based on complete 16S rRNA sequences.


 
Formulation development
 
Powder and granular based formulations for facultative chemolithoautotrophic sulfur oxidizing bacteria were developed using lignite, compost and clay soil (Fig 6).

​

Fig 6: Various formulations of facultative chemolithoautotrophic sulphur oxidizing bacteria.


       
Sulfur is cycled biogeochemically by microorganisms (Friedrich et al., 2005). Sulfur-oxidizing bacteria, including chemolithotrophs and chemoheterotrophs, utilize thiosulfate. It is most suitable for the investigation of sulfur lithotrophic process (Mukhopadhayaya et al., 2000). In the Present study, 7 chemolithoautotrophic and 5 facultative chemolithoautotrophic sulfur oxidising bacteria were isolated from different rice ecosystems such as wetland, aerobic and SRI. In an earlier study, Starkey (1935) isolated sulfur-oxidising bacteria from black clay loams. He also reported that the characteristics of growth were diverse; in some cases, the medium was turbid. A low level of sulfur oxidizing bacteria is found in alkali soils.An alkali soil enriched with appropriate bacteria resembles the A. thiooxidans and S. novella bacterial species. From Galapagos hydrothermal vent, obligate heterotrophic sulfur oxidizers were repeatedly isolated that presumably oxidized thiosulfate either to sulfate (acid producing Thiobacillus like) or to polythionates (base producing Pseudomonas) (Vidyalakshmi and Sridar, 2013). Ito et al., (2005) isolated bacteria from wastewater biofilms that oxidize sulfur aerobically.
       
Sulfur oxidizing bacteria such as Bosea thiooxidans, Paracoccus thiocyanatus, Pseudaminobacter salicylatoxidans, Paracoccus pantotrophus, Paracoccus bengalensis, Tetrathiobacter kashmirensis and Mesorhizobium thiogangeticum,  were isolated from rhizosphere and bulk soils of agricultural fields of India (Ghosh et al., 2005; 2006; Das et al., 1996; Ghosh and Roy, 2006a, b, 2007; Deb et al., 2004). Recently, Anandham  et al., (2005; 2007; 2008a; 2009; 2010) have been documented that the rhizosphere soils of crop plants in Korea are dominated by both obligate and facultative chemolithotrophic thiosulfate oxidizing bacteria (Yim et al., 2008). Isolated sulfur oxidizing bacteria were screened based on pH reduction and titrable acidity. Similarly, earlier study, Anandham  et al., (2005; 2007) screened sulfur oxidizing bacteria based on pH reduction test. Vassilev et al., (2001) adopted titrable acidity as one of the criteria to screen phosphate solubilizing bacteria. pH reduction and titrable acidity are indication of indirect oxidation of sulfur oxidation. Sulfur oxidizing bacteria oxidize thiosulfate into sulfuric acid which was responsible for reduction of pH of the medium (Anandham et al., 2007; Chaudhary et al., 2022). All the isolated sulfur oxidizing bacteria in the present investigation are Gram negative rods except Bacillus tequilensis S2Y2- a is Gram positive. In a previous study, Anandham et al., (2008b) reported the presence of sulfur oxidation traits in Gram positive Microbacterium phyllosphaerae and Leifsonia shinshuensis. In the current investigation., isolated facultative chemo lithoautotrophic sulfur oxidizing bacteria exhibited different nutritional ability. In a previous study, facultative chemo lithoautotrophic Pandoraea thiooxydans, Burkholderia kururiensis subsp. thiooxydans, Dyella thiooxydans exhibited autotrophic, heterotrophic abilities (Anandham  et al., 2009; 2010; 2011).
       
To best of our knowledge this is the first study to report the presence of sulfur oxidation trait in Pseudomonas beteli, Providencia stuartii and Bacillus tequilensis. Tetrathiobacter kashmirensis reclassified as Advenella kashmirensis was originally recovered in in garden soils of Kashmir, the same strain also isolated from paddy soil of present study (Ghosh et al., 2005). It was attributed that selective pressure (sulfur) used by Ghsoh et al., (2005) same selective pressure was also used in this study.

CONCLUSION

Totally five facultative chemolithoautotrophic sulfur oxidizing bacteria were autotrophic and chemo heterotrophic in nature recovered from different rice ecosystems which efficiently utilizes inorganic sulfur compounds as electron donors to generate energy. This investigation is widely distributed in isolated genera namely, Pseudomonas beteli, Advenella kasmirensis, Pseudomonas beteli, Providencia stuartii and Bacillus tequilensis. The current research explores the presence of facultative chemolithoautotrophs in rice ecosystems, which have a great ability to lower the pH of the culture medium and effectively create sulfate ion. The pH lowering ability of SOB allows them to be used for alkali soil restoration and phosphate solubilisation in the future.

ACKNOWLEDGEMENT

The authors thank the Department of Agricultural Microbiology, Agricultural College and Research Institute, Madurai, for providing all facilities for conducting this study.
 
Funding
 
This work is self-supported.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 

CONFLICT OF INTEREST

     The authors declare no competing interests.

REFERENCES


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    Full Research Article

    Delineation of Sulphur Bacteria Isolated from Rice Rhizosphere Soil

    K
    K. Sowmya1,*
    S
    S.L. Sivapriya2
    1Department of Agricultural Microbiology, School of Agricultural Sciences, Bharath Institute of Higher Education and Research, Tambaram, Chennai-600 001, Tamil Nadu, India.
    2Janani Public School, Bangalore-560 001, Karnataka, India.

    ABSTRACT

    Background: Crop yields are greatly reduced by sulfur-deficient conditions, as sulfur is an essential nutrient for plant growth. So, sulfur fertilizers are generally added to soils to alleviate this deficiency, usually in a reduced form, such as elemental sulfur. Despite this, microorganisms are required to oxidize reduced sulfur fertilizers into sulfate before they can be absorbed by plants. Recently, sulfur deficiency was reported widely in rice. Till date there are no suitable bio-inoculants for rice. Hence, the present study was aimed to develop a sulfur bio-inoculant for rice.

    Methods: In this present investigation, totally 9 chemolithoautotropic sulfur oxidizing bacteria (3 from aerobic, 4 from wet land and 2 from SRI systems) were isolated from various rice ecosystems. Chemolithoautotrophic strain S1-3 isolated from SRI system and strain W3-1 isolated from wet land were found to efficient based on pH reduction and titrable acidity. Totally five facultative chemo lithoautotrophic sulfur oxidizing bacteria (two from wet land and 3 aerobic samples) were recovered from different rice ecosystems.

    Result: Facultative chemolithoautotrophic strain S1Y1-bLL and S2Y2-b significantly reduced the medium pH (4.3) and consumed the maximum alkali 1.7 and 1.8 mL and found to be efficient. All the isolated facultative chemolithoautotrophic sulfur were autotrophic and chemoheterotrophic in nature. Molecular characterization study revealed that strains S1Y2 - aS, S1Y1 - Bll, S2Y2 - b, S2SA - Sa and S2Y2 - an exhibited 99.5, 99.7, 99.5, 99.6 and 99.9% sequence similarities to Pseudomonas beteli, Advenella kasmirensis, Pseudomonas beteli, Providencia stuartii and Bacillus tequilensis, respectively. Further, developed lignite and compost based and clay pellet formulation for facultative chemolithoautotrophic sulfur oxidizing bacteria. Yet, plant inoculations studies should be carried out for isolated sulfur oxidizing bacteria from this study before recommended to the farmers.

    INTRODUCTION

    In addition to nitrogen, phosphorus and potassium, sulfur (S) is becoming increasingly recognized as a major plant nutrient. A compound containing reduced sulfur is produced from the amino acids cysteine and methionine, which act as precursors (Narayan et al., 2023). In addition to holding structural and functional roles within macrobiomolecules, sulfur modulates many physiological processes and makes plants more tolerant to abiotic stress (Haneklaus et al., 2003). Plants and animals require sulfur to survive. A sulfur deficiency in the field was first reported in 1933, despite plants’ need for sulfur in comparable amounts to phosphorus. The first report of sulfur deficiency in wetland rice was made in 1938. Dry-land crops as well as wetland rice have been found to be affected by sulfur deficiency over the last 10 years. The tropics are generally characterized by sulfur deficiency in Andosols, Vertisols, Alfisols, Ultisols and Oxisols. Several Asian countries, including Bangladesh, Burma, India, Indonesia, Japan, Philippines and Sri Lanka, have reported sulfur deficiency of wetland rice (Roy et al., 2017). Twenty-three crops have been reported to respond to sulfur in forty tropical countries (Jenni, 2020). A decrease in organic manures, intensive cropping and decreased atmospheric deposition have contributed to this increase in sulfur deficiencies (Dawar et al., 2023). The use of sulfur fertilizers is invariably used to alleviate sulfur deficiencies in soils, usually in reduced forms, such as elemental sulfur. Use of S oxidizers enhances the rate of natural oxidation of S and speed up the production of sulfates and makes them available to plants at their critical stages, consequently resulting in increased plant yield (Anandham et al., 2008a, b). Micororganisms contribute the biogeochemical cycling of sulfur (Friedrich et al., 2005). Various microorganisms (prokaryotes, green sulfur bacteria) have been described which utilize sulfur compounds obligately or facultatively as electron donors and oxidize them to sulfate. Sulfur oxidizing bacteria such as Bosea thiooxidans, Paracoccus thiocyanatus, Pseudamino- bacter salicylatoxidans, Paracoccus pantotrophus, Paracoccus bengalensis, Tetrathiobacter kashmirensis and Mesorhizobium thiogangeticum were isolated from several ecological niches (Sowmya and Sivapriya 2026).

    MATERIALS AND METHODS

    Rhizosphere sample collection
     
    Rhizosphere soil samples were collected from rice cultivated land at various ecosystems (Wetland, SRI and aerobic) (Table 1). Three plants at flowering stages were selected and removed from the soil, placed in a polythene bag, immediately transported to the laboratory and processed. The plants were shaken gently to separate the soil that was not tightly adhering to the roots. The rhizosphere soil (soil attached to the roots after gentle shaking) and fine roots (approximately 1 cm length) were collected from the plants and used for enrichment isolation (Anandham et al., 2008).

    Table 1: Plant source for isolation of sulfur oxidizing bacteria.


     
    Enrichment Isolation
     
    Enrichment isolation of chemolithoautotrophic sulfur-oxidising bacteria
     
    For isolation of chemolithoautotrophic sulfur oxidizing bacteria, three 1 g replicates of each rhizosphere soil samples were added into 10 mL of liquid mineral salts thiosulfate (MST) medium containing (g L-1) NH4CI, 1.0; K2HPO4, 4.0; KH2PO4, 1.5; MgSO4.7H20, 0.5; Na2S2O3.5H20, 5.0; yeast extract, 0.05; bromocresol purple, 0.002; trace element solution 5 ml; pH 7.5). The medium was then incubated at 30oC in the dark to avoid growth of phototrophic bacteria on a rotary shaker (120 rpm) until the color of the bromocresol purple changed to yellow.
     
    Enrichment isolation of facultative chemolithoautotrophic sulfur-oxidizing bacteria
     
    For isolation of chemolithoautotrophic sulfur-oxidizing bacteria, three I g replicates of each rhizosphere soil sample were added into 10 mL of liquid mineral salts thiosulfate yeast extract (MSTYE) medium containing (g L-l) NH4CI, 1.0; K2HPO4, 4.0; KH2PO4, 1.5; MgSO4.7H20, 0.5; Na2S2O3.5H20, 5.0; yeast extract, 5.0; bromocresol purple, 0.002; trace element solution 5 ml; pH 7.5). The medium was then incubated at 30oC in the dark to avoid growth of phototrophic bacteria on a rotary shaker (120 rpm) until the colour of the bromocresol purple changed to yellow.
     
    Purification of sulfur-oxidising bacteria
     
    For isolation of pure cultures, streak plate method is followed a Loop full of culture is streaked on the surface of MST/MSTYE agar plates (Fig 6). The colonies that developed yellow halo against purple background, indicative of the production of sulfuric acid resulting from the oxidation of thiosulfate were picked and streaked on solid MST medium until uniform colony morphology was observed. Colonies were transferred 3 times to be considered as pure, in addition, the purity of the strains was checked microscopically. The pure bacterial strains were maintained on MST agar plates, sub cultured every week and subjected to further studies.

    Screening of efficient sulfur-oxidizing bacteria
     
    pH reduction test
     
    Isolated chemolithoautotrophic and facultative chemolitho-autotrophic sulfur-oxidising bacteria were inoculated in MST and MSTYE broth and incubated for 5 days at 30oC. The End of sulfur oxidation is the production of sulfuric acid; hence, the pH of the medium would be reduced. The reduction in pH was recorded using the pH meter (Medox, MX-1292-01, India) at 25oC.
     
    Titrable acidity
     
    One mL of stationary phase cultures of chemolitho-autotrophic and facultative chemolithoautotrophic sulfur-oxidising bacteria cultivated in liquid MST and MSTYE were collected in a beaker and drops of 0.1% phenolphthalein were added. At this moment, it is colourless. It was titrated against 0.1N NaOH until the appearance of pink colour, which indicates the end of the titration. Titrable acidity indicates the indirect production of acid.
     
    Morphological characterization
     
    Phenotypic characters such as colony colour, colony diameter, cell shape and Gram staining were performed as per the standard procedure (Smibert and Krieg, 1994).
     
    Nutritional characterization of facultative chemoltiho-autotrophic sulfur oxidizing bacteria
     
    Facultative cheomolithoautotrophic sulfur oxidizing bacteria were streaked on MSTA (Mineral salts thiosulfate agar), NA (Nutrient agar), TSA (tryptic soy agar) and LBA (Luria Bertani agar) and incubated at 30oC for 5 d then growth was checked.
     
    Molecular characterization and phylogenetic analysis
     
    The purified bacteria were grown in MST broth and harvested in the log to initial stationary phase. The genomic DNA was extracted using standard methods described (Wright et al., 2017). The genomic DNA was amplified using 16S rRNA universal primer (27f: 5' -AGA GTT TGA TCC TGG CTC AG-3' and 1492r: 5' GGT TAC CTT GTT ACG ACT T-3' ) in a thermocycler (Veriti™ 96-Well Fast Thermal Cycler). The authentication potential of SOB was verified by 16S rRNA amplification followed by sequencing for identification. The obtained sequences were performed with a similarity search using BLAST. A phylogenetic tree was constructed with existing 16S rRNA sequences of SOB from different eubacteria obtained from NCBI GenBank database.
     
    Development of formulation
     
    Development of powdered formulation
     
    Facultative chemolithoautotrophic sulfur oxidizing bacteria grown in nutrient broth were used for formulation development. One kg of powdered, neutralized and/or sterilized lignite and compost were mixed with stationary phase culture (1 x 109 cfu mL-1) under aseptic condition, allowed for 2d and packed.
     
    Development of powdered formulation
     
    One Kg of clay soil was powdered and sterilized and cooled. Then clay soil was mixed with 400 mL of stationary phase Facultative chemolithoautotrophic sulfur oxidizing bacterial culture (1 x 109 cfu mL-1) and pelletized with hand pelletizer and air dried further cut into approximately 1 cm size and stored.

    RESULTS AND DISCUSSION

    Isolation of chemolithoautotrophic sulfur-oxidising bacteria
     
    In total, 27 rice rhizosphere soil samples were collected from various rice ecosystems, such as aerobic, wetland and SRI rice, of which 25 samples contained the chemolitho- autotrophic sulfur-oxidising bacteria. It was inferred that the change of colour from purple to yellow, which indicated the end of sulfur oxidation, is the production of sulfuric acid, ultimately reducing the pH of the medium, in turn changing the medium colour from purple to yellow (Table 2). Above 25 positive samples, the isolates that showed good growth (3 from aerobic, 4 from wetland and 2 from SRI systems) were taken for purification (Table 3).

    Table 2: Isolation of chemolithoautotropic sulfur oxidizing bacteria from different ecosystems.



    ​

    Table 3: Selection of chemolithoautotrophic sulfur oxidizing bacteria for purification.


     
    Screening of chemolithoautotrophic sulfur-oxidising bacteria
     
    Efficient chemolithoautotrophic sulfur-oxidising bacteria were screened based on pH reduction and Titrable acidity, indirectly indicating sulfur oxidation. Among isolates, strain S1-3 isolated from the SRI system showed the maximum reduction of pH (5.4), followed by the wetland strain W3-1 (5.6). Strains W3-1 and S1-3 consumed the maximum alkali 2 and 1.2 mL, respectively (Table 4).

    ​

    Table 4: Screening of chemolithoautotrophic sulfur oxidizing bacteria based on pH reduction and titrable acidity.


     
    Isolation of facultative chemolithoautotrophic sulfur-oxidising bacteria
     
    In total, 27 rice rhizosphere soil samples were collected from various rice ecosystems, such as aerobic, wetland and SRI rice. Of which 5 samples (two wetlands and 3 aerobic samples) contained the efficient facultative chemolithoautotrophic sulfur-oxidising bacteria (Table 5). Above 5 positive samples, 5 facultative chemolithoautotrophic sulfur-oxidising bacteria were recovered (Table 5; Fig 1).

    ​

    Table 5: Isolation of facultative chemolithoautotrophic sulfur oxidizing bacteria.



    Fig 1: Purification of facultative chemolithoautotophic Sulfur oxidizing bacteria.


     
    Screening of facultative chemolithoautotrophic sulfur-oxidising bacteria
     
    Efficient facultative chemolithoautotrophic sulfur-oxidising bacteria were screened based on pH reduction and titrable acidity, indirectly indicating sulfur oxidation. Strain S1Y1-bLL and S2Y2-b significantly reduced the medium pH (4.3) and consumed the maximum alkali 1.7 and 1.8 mL (Table 6).

    ​

    Table 6: Screening of facultative chemolithoautotrophic sulfur oxidizing bacteria based on pH reduction and titrable acidity.


     
    Morphological characterisation of facultative chemolitho-autotrophic sulfur-oxidising bacteria
     
    Among the screened facultative chemolithoautotrophic sulfur-oxidising bacteria, all were Gram-negative rods except strain S2Y2-a, which was Gram-positive (Table 7).

    ​

    Table 7: Morphological characterization of facultative chemo lithoautotrophic sulfur oxidizing bacteria.


     
    Nutritional characterisation of facultative chemolitho-autotrophic sulfur- oxidising bacteria
     
    All the isolated facultative chemolithoautotrophic sulfur-oxidising bacteria strains were able to grow on autotrophic medium (MSTA) as well as on NA, TSA and LBA. This indicated that all the facultative chemolithoautotrophic sulfur oxidizing bacteria isolated from this study were autotrophic as well as chemoheterotrophic in nature (Table 8; Fig 2).

    ​

    Table 8: Nutritional characterization of facultative chemo lithoautotrophic sulfur oxidizing bacteria.



    Fig 2: Growth of facultative chemolithoautotrophic sulphur oxidizing bacteria in NA medium.


     
    Molecular characterization of facultative chemolitho-autotrophic sulfur oxidizing bacteria
     
    Molecular characterization of sulfur oxidizing bacteria was performed through 16S rDNA sequencing. Sequence results revealed that strains SlY2- as, S1Y1-bLL, S2Y2-b. S2SASa and S2Y2- a cxhibited 99.5, 99.7, 99.5, 99.6 and 99.9% sequence similarties to Pseudomonas beteli, Advenella kashmirensis, Pseudomonas beteli Providencia stuartii and Bacillus tequilensis, respectively. In phylognelic analyses revealed that clustering patern were similar in both neighbor-joining and maximum likely hood trees. Irrespective of phylogenetic methods used, strain S1Y 1- bLL formed cluster with Advenella kashmirensis which was supported by bootstrap value of 100, 92 and 9 in neighbor -joining (NI), maxmimum-likely hood (ML) and maximum- parsimony (MP) trees. Similarly, strains S1Y2- aS and S2Y2- b clustered with Pseudomonas beteli. Strains S2SA -Sa and S2Y2 formed separate clade with Providencia stuartiiand Bacillus tequilensis, respectively (Table 9; Fig 3, 4, 5).

    ​

    Table 9: Molecular characterization of facultative chemolithoautotrophic sulfur oxidizing bacteria isolated from different ecosystems.



    ​

    Fig 3: Neighbour-joining phylogenetic tree based on complete 16S rRNA sequences.



    ​

    Fig 4: Maximum likely hood phylogenetic tree based on complete 16S rRNA sequences.



    ​

    Fig 5: Maximum parsimony phylogenetic tree based on complete 16S rRNA sequences.


     
    Formulation development
     
    Powder and granular based formulations for facultative chemolithoautotrophic sulfur oxidizing bacteria were developed using lignite, compost and clay soil (Fig 6).

    ​

    Fig 6: Various formulations of facultative chemolithoautotrophic sulphur oxidizing bacteria.


           
    Sulfur is cycled biogeochemically by microorganisms (Friedrich et al., 2005). Sulfur-oxidizing bacteria, including chemolithotrophs and chemoheterotrophs, utilize thiosulfate. It is most suitable for the investigation of sulfur lithotrophic process (Mukhopadhayaya et al., 2000). In the Present study, 7 chemolithoautotrophic and 5 facultative chemolithoautotrophic sulfur oxidising bacteria were isolated from different rice ecosystems such as wetland, aerobic and SRI. In an earlier study, Starkey (1935) isolated sulfur-oxidising bacteria from black clay loams. He also reported that the characteristics of growth were diverse; in some cases, the medium was turbid. A low level of sulfur oxidizing bacteria is found in alkali soils.An alkali soil enriched with appropriate bacteria resembles the A. thiooxidans and S. novella bacterial species. From Galapagos hydrothermal vent, obligate heterotrophic sulfur oxidizers were repeatedly isolated that presumably oxidized thiosulfate either to sulfate (acid producing Thiobacillus like) or to polythionates (base producing Pseudomonas) (Vidyalakshmi and Sridar, 2013). Ito et al., (2005) isolated bacteria from wastewater biofilms that oxidize sulfur aerobically.
           
    Sulfur oxidizing bacteria such as Bosea thiooxidans, Paracoccus thiocyanatus, Pseudaminobacter salicylatoxidans, Paracoccus pantotrophus, Paracoccus bengalensis, Tetrathiobacter kashmirensis and Mesorhizobium thiogangeticum,  were isolated from rhizosphere and bulk soils of agricultural fields of India (Ghosh et al., 2005; 2006; Das et al., 1996; Ghosh and Roy, 2006a, b, 2007; Deb et al., 2004). Recently, Anandham  et al., (2005; 2007; 2008a; 2009; 2010) have been documented that the rhizosphere soils of crop plants in Korea are dominated by both obligate and facultative chemolithotrophic thiosulfate oxidizing bacteria (Yim et al., 2008). Isolated sulfur oxidizing bacteria were screened based on pH reduction and titrable acidity. Similarly, earlier study, Anandham  et al., (2005; 2007) screened sulfur oxidizing bacteria based on pH reduction test. Vassilev et al., (2001) adopted titrable acidity as one of the criteria to screen phosphate solubilizing bacteria. pH reduction and titrable acidity are indication of indirect oxidation of sulfur oxidation. Sulfur oxidizing bacteria oxidize thiosulfate into sulfuric acid which was responsible for reduction of pH of the medium (Anandham et al., 2007; Chaudhary et al., 2022). All the isolated sulfur oxidizing bacteria in the present investigation are Gram negative rods except Bacillus tequilensis S2Y2- a is Gram positive. In a previous study, Anandham et al., (2008b) reported the presence of sulfur oxidation traits in Gram positive Microbacterium phyllosphaerae and Leifsonia shinshuensis. In the current investigation., isolated facultative chemo lithoautotrophic sulfur oxidizing bacteria exhibited different nutritional ability. In a previous study, facultative chemo lithoautotrophic Pandoraea thiooxydans, Burkholderia kururiensis subsp. thiooxydans, Dyella thiooxydans exhibited autotrophic, heterotrophic abilities (Anandham  et al., 2009; 2010; 2011).
           
    To best of our knowledge this is the first study to report the presence of sulfur oxidation trait in Pseudomonas beteli, Providencia stuartii and Bacillus tequilensis. Tetrathiobacter kashmirensis reclassified as Advenella kashmirensis was originally recovered in in garden soils of Kashmir, the same strain also isolated from paddy soil of present study (Ghosh et al., 2005). It was attributed that selective pressure (sulfur) used by Ghsoh et al., (2005) same selective pressure was also used in this study.

    CONCLUSION

    Totally five facultative chemolithoautotrophic sulfur oxidizing bacteria were autotrophic and chemo heterotrophic in nature recovered from different rice ecosystems which efficiently utilizes inorganic sulfur compounds as electron donors to generate energy. This investigation is widely distributed in isolated genera namely, Pseudomonas beteli, Advenella kasmirensis, Pseudomonas beteli, Providencia stuartii and Bacillus tequilensis. The current research explores the presence of facultative chemolithoautotrophs in rice ecosystems, which have a great ability to lower the pH of the culture medium and effectively create sulfate ion. The pH lowering ability of SOB allows them to be used for alkali soil restoration and phosphate solubilisation in the future.

    ACKNOWLEDGEMENT

    The authors thank the Department of Agricultural Microbiology, Agricultural College and Research Institute, Madurai, for providing all facilities for conducting this study.
     
    Funding
     
    This work is self-supported.
     
    Disclaimers
     
    The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
     

    CONFLICT OF INTEREST

         The authors declare no competing interests.

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