JOURNAL OF CULTURE COLLECTIONS

Vo= lume 5, 2006-2007, pp. 73-77

ISOLATION= AND CHARACTERIZATION OF

SULPHUR OXIDIZING BACTERIA

Rajagopal Vidyalakshmi^1*and R. Sridar²

¹Paddy Processing Research Cen= tre, Thanjavur-4, Tamil Nadu, India;

²Department of Microbiology, T= amil Nadu Agricultural University, Coimbatore, = Tamil Nadu, India

^*Corresponding author, e-mail: onlyvidu@yahoo.co.in

Summary

Sulphur oxidizing bacteria were isolated from different samples viz., paddy rhizosphere, pulse rhizosphere, sewage, biogas slurry, tannery effluent and mine soil. Out of the 28 isolates obtained, 14 were screened based on their efficacy to reduce the pH of the growth medium from 8.0 to = £ 5.0. The selected isolates were characterized and related to the genus Thiobacillus.

Key words: sulphur oxidizing bacteria, Thiobacillus, sulphate.

Introduction

Sulph= ur is now considered the fourth major plant nutrient after N, P = and K, and is one of the sixteen nutrient elements which are essential for the gro= wth and development of plants, especially in the agricultural crop production. = This is mainly because of its wide-spread deficiency in the soil world over. The importance of sulphur is equal to that of nitrogen in terms of protein synthesis, and in terms of crop uptake it exceeds even that of phosphorus. = The majority of sulphur taken up by plant roots is in the form of sulphate (SO<= sub>4), which undergoes a series of transformations prior to its incorporation into= the original com-pounds [10].

The soil microbial biomass is the k= ey driving force behind all sulphur transformation. The biomass acts as both a source and sink for inorganic sulphate. The latter make sulphate available = from element sulphur or any reduced forms of sulphur through its oxidation proce= ss in the soil. The role of chemolithotrophic bacteria of the genus Thiobacillus in this process is essential. The objective of this study was to isolate and characterize the sulphur oxidizing bacteria from various sources.

Materials and Methods

Isolation of sulphur= oxidizing bacteria. Isolation was performed using samples collected from seven differe= nt sources viz., paddy rhizosphere soil (Wetland, Tamil Nadu Agricultural University, Coimbatore), lab rhizosphere soil (Millet Breeding Station, Tam= il Nadu Agricultural University, Coimbatore), black gram rhizosphere soil (Far= mers field, Pondicherry), sewage water (Department of Bioenergy, Tamil Nadu Agricultural University, Coimbatore), tannery effluent (Department of Environmental Sciences, Tamil Nadu Agricultural University, Coimbatore) and mine soil (Neyveli Lignite Corporation, Neyveli).

Microbiological media. The med= ia employed for the isolation of sulphur oxidizing bacteria include Starkey br= oth [13] composed of 3.0 g KH₂PO₄, 0.2 g MgSO4×7H₂O, 0.2 g CaCl₂×2H₂O, 0.5 g (NH₄)2SO₄, traces of FeSO₄ in 1000 ml distilled water with pH 8.0; NCL broth [15] consisted of 0.2 g (NH₄)₂SO₄, 0.5 g MgSO₄×7H₂O, 0.25 g CaCl₂ 2= H₂O, traces of FeSO₄, in 1000 ml of distilled water with pH 3-5; and = the thiosulphate broth [2] contained 5.0 g Na₂S₂O3, 0.1 g K₂HPO₄, 0.2 g NaHCO₃, 0.1 g NH₄Cl in 1000 ml distilled water, with pH 8.0. B= romo cresol purple was the indicator used. For isolation of heterotrophic oxidiz= ers 5.0 g of glucose per litre of Starkey, NCL and Thiosulphate broth was added. Elemental sulphur at 10 g per litre was added to Starkey and NCL broths and half-an-hour steam steril= ized for three consecutive days. Thiosulphate broth was devoid of S⁰.= One gram or one ml of the sample was added to 20 ml of the broth dispensed in tubes, under aseptic conditions. The tubes were incubated in BOD incubator = at 32 ^oC for 25 days. The isolates obtained were purified by transferring to fresh broth thrice at fortnightly intervals. The isolates w= ere then streaked on thiosulphate agar medium and individual colonies were obtained. The single colonies were pick-ed and preserved on thiosulphate slants. The pure cultures were labelled and used for characterization and further studies.

Screening = of isolates by pH reduction test. The obtained is= olates were inoculated in the growth media with initial pH adjusted to 8.0 and incubated at 32 ^oC for 15 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 5.0 or less than 5.0. The selected isolates were further studied for their morphology, Gram reaction, colony characters and nutritional type.

Cell and = colony morphology. Negative staining was done and the morphology of the isolates was studied under a microscope. Gram staining [7= ] of the isolates was performed. For colony characterization Starkey agar and thiosulphate agar media were prepared with pH adjusted to 8.0. The isolates were plated in sterile Petri dishes by the pour plate method and the plates incubated at 32 ^oC for 15 days. Colony characters were obse= rved after the incubation period.

Utilizati= on of sulphur sources. The isolates were studied fo= r the utilization of elemental sulphur and thiosulphate broth. They were inoculat= ed in both the sterilized broths (initial pH 6.0) and incubated in the BOD incubator for 15 days. The growth was assessed by pH reduction and microsco= pic observation.=

Selection of s= uitable media for autotrophic and heterotrophic sulphur oxidizing bacteria. Starkey broth with initial pH o= f 8.0 was prepared with and without glucose. The isolates were inoculated and incubat= ed at 32 ^oC for 15 days. Based on the pH reduction of the broth with and without glucose, the isolates were classified as chemoheterotrophs= and chemo-autotrophs. Heterotrophic isolates were also inoculated in NCL and thiosulphate broth prepared with glucose. Autotrophic isolates were inocula= ted in the same broths prepared without glucose. Inoculated tubes were incubate= d at 32 ^oC for 15 days. Control tubes and three replications were maintained. The pH of the broth was recorded after the incubation period.

Influence of biotin on the elemental sulphur oxida= tion. Filter sterilized biotin solutio= n (0.1 per cent) was added to the Starkey broth. The pH reduction was measured at = 24 hour intervals from the tenth up to the 15^th day of incubation u= sing a pH meter. Simultaneously, a control was maintained.

Growth of type culture. The strain Th= iobacillus thiooxidans MTCC 468 was obtained from the Microbial Type Culture Collection (MTCC), Chandigarh= , Haryana and was grown in the medium as recommended by the MTCC and used as a reference culture.

Results

Isolation and screen= ing of sulphur oxidizing bacteria

A total of twenty eight isolates were obtained fro= m seven different samples. Among them, 14 isolates were selected based on the pH reduction test (Fig. 1) and named as follows: SR, AP2, AN, NP2, NS, TP2, TB, TR, ASE, ATE, ATR, ASS and ATB. Among the 14 isolates obtained, the isolate from the black gram rhizosphere soil (TP2) reduced the pH to 4.5, 5= .5 and 4.0 (control 8.0) of the Starkey, NCL and thiosulphate broth respective= ly, with-in 12 days. The isolate obtained from paddy rhizosphere soil (SR) caus= ed a pH reduction of 4.5 and 6.5 (control 8.0) in Starkey and thiosulphate broth respectively, within 10-15 days and no reduction was found in NCL broth. The results are furnished in Table1. The isolate NP2 reduced the pH to 5.0 of Starkey, NCL and thiosulphate broths. The isolates SN, NS, TR, ATE and ATB = did not show a remarkable reduction in pH of the growth media.

Fig.1. Reduction of pH in the growth media by sulphur oxidizing bacteria.<= o:p>

Table 1. Reduction of pH in the growth media by sulphur oxidiz= ing bacteria (SOB).

Name of SOB isolate

pH Reduction in growth media

Starkey broth

NCL broth

Thiosulphate broth

SR

4.5

8.0

6.5

SP2

4.5

8.0

5.0

SN

6.5

8.0

8.0

SS

5.5

5.5

8.0

NP2

5.0

5.0

5.0

NS

8.0

5.5

6.0

TP2

4.5

5.5

4.0

TB

5.5

5.5

8.0

TR

8.0

8.0

6.0

ASE

8.0

6.0

4.5

ATE

8.0

6.5

8.0

ATR

8.0

6.0

5.5

ASS

6.0

5.5

6.0

ATB

6.0

6.0

8.0

= ;

Characterization of = the selected isolates

The results of the isolates characterization are presented in = Table 2 and Fig. 2. Invariably, all the organisms were short rods and gram negati= ve, but differed in the utilization of sulphur sources.

Table 2. Characterization of the screened isolates.

Isolates

Morphology

Gram reaction

Utilization

Influence of biotin

Colony character

Nutritional type

Elemental sulphur

Thio-sulphate

Glucose

SR

Short rods

Negative

+

+

+

+

Smooth, round straw yellow colour colonies

Hetero-
troph

SP2

Short rods

Negative

+

+

+

+

SN

Short rods

Negative

+

-

+

+

SS

Short rods

Negative

+

-

+

+

NP2

Short rods

Negative

+

+

+

+

NS

Short rods

Negative

+

+

+

+

TP2

Short rods

Negative

+

+

+

+

TB

Short rods

Negative

+

+

+

+

TR

Short rods

Negative

-

+

+

+

ASE

Short rods

Negative

+

+

-

+

Smooth, raised pink colour colonies

Auto-
troph

ATE

Short rods

Negative

-

+

-

+

ATR

Short rods

Negative

-

+

-

+

ASS

Short rods

Negative

+

+

-

+

ATB

Short rods

Negative

+

+

-

+

Legend: utilized (+), unutilized (-).

Fig. 2. Growth of sulphur oxidizing bacteria on thiosulphate agar medium.

The isolates SN and SS utilized only S⁰, whereas TR= , ATE and ATR utilized thiosulphate only. The isolates SR, SP2, NP2, NS, TP2, TB,= ASE, ASS and ATB utilized both S⁰and thiosulphate. The heterotrophic isolates utilized glucose, whereas the autotrophic isolates did not utilize glucose. All the isolates were positively influenced by biotin. The heterotrophic colonies were smooth, round straw yellow coloured, and autotrophic colonies were smooth, raised, pink coloured.

&= nbsp;

Discussion

Twenty-eight isolates were obtained from different sources viz., soil, sewage, biogas slurry,= mine soil, tannery effluent etc. Earlier studies on isolation of sulphur oxidizi= ng bacteria by various researchers reveal their existence in mud soil, canal water, other fresh water sources [11] and acid streams [9]. T. ferrooxidans and other Thiobacillus sp. were isolated from uranium mines by Deborah Berthelot [3]. A similar organism was also reported from garden soil, activated sludge [1] and soil sulphur com-post [4]. Dave = and Upadhyay isolated thiosulphate oxidizing organisms from thermal springs [5]. Heterotrophic isolates of thiosulphate producing bacteria, which acidified = the medium moderately by ca.0.5 to 1.0 pH units, were obtained from marine sediments and hydrothermal vents [10].

Out of the 28 isolates 14 were selected based on pH reduction (initial 8.0, final £ 5.0 in 15 days)= . The pH reduction of the medium was due to the production of sulphuric acid. Reduct= ion in pH of the growth medium by sulphur oxidizing bacteria was reported by Do= nati [6]. The selected isolates were characterized to be short rods, Gram negati= ve, utilizing biotin and one or more of the sulphur sources. The heterotrophic colonies were smooth, round and straw yellow probably due to the sulphur deposition. Similar results were reported by several workers [8, 11, 16] who isolated from soil compost sulphur oxidizing bacteria which were short rods= and morphologically similar to Thiobaci= llus. The organisms reduced the pH from 5.6-6.2 to 2.6-2.8 and utilized both elemental sulphur and thiosulphate [16]. All the autotrophic and heterotrop= hic isolates were positively influenced by biotin, which was in conformity with= the findings of Kelly and Harrison [11]. Based on the morphological and physiological studies and on comparison with the reference culture T. thiooxidans MTCC 468, all the 14 isolates were confirmed to belong to the genus Thiobacillus.

The present study emphasizes the importance and the role of sulphur oxidizing bacteria in the oxidation of sulphur in soil. These Thiobacillus isolates can be incorporated to enhance sulphur oxidation in soil and to increase soil available sulphate. Also, the pH reducing property of sulphur oxidizing bacteria by the product= ion of sulphuric acid can be utilized for reclamation of alkali soils. <= /p>

References

1. Ayyar, C. V. R., T. S. S. Perumal, R. V. Norris, 1929. J. Indian Inst. Sci., 11, 85-90.

2. Beijerinck, M. W., 1904. = Arch. Sci. Exactes Nat. Haarlem., Ser.<= /span> 2, 9131-9157.

3. Berthelot, L. D., G. Ledu= ec, G. Ferroni, 1992. Canadian J. Microbiol., 39, 384= -388.

4. Brown, H. D., 1923. J. Amer. Soc. Agron., 15, 350-382.

5. Dave, S. R., N. M. Upadhy= ay, 1993. Indian J. Micorbiol., 33, 241-244.

6. Donati, E., G. Curutchet,= C. Pogliani, P. Tedesco, 1996. Pr= ocess Biochem., 31, 129-1= 34.

7. Gram, H.C, 1884. Fortschritte Medizin, 2, 185-89.

8. Izumikubo, I. K., T. Takeuchi, M. Furusawa, Y. Arikawa, T. Kanagawa, 1995. Can. J. Microbiol., 41, 366-371.

9. Johnson, D. B., M. A. Gha= uri, M. F. Said, 1992. Appl. Enviro= n. Microbiol., 58, 1423-1428.

10. Katyal, J. L., K. L. Shar= ma, K. Srinivas, 1997. ISI/FAI/IFA Symposi= um on sulphur in balanced fertilization, 13-14 Feb., <= st1:City w:st=3D"on">New Delhi, India, Proceedings, 2/1-2/11.

11. Kelly, D. P., A. P. Harri= son, 1988. Genus Thiobacillus Beijer= inck. In: Bergey’s manual of Systamatic Bacteriology= , G. T. Staley, N. Pfenning, J. G. Holt (Eds.), Vol. 3, Baltimore: Willi= ams & Wilkinson Co., 1842-1871.

12. Starkey, R. L, 1935. <= span style=3D'mso-bidi-font-weight:bold'>Soil Sci., 39, 197-219.

13. Starkey, R. L., V. G. Col= lins, 1923. Autotrophs. In: Methods in Microbiology, J. = R. Norris, D. W. Ribbons (Eds), = New York: Academic Press, 38, 55-73.

14. Teske, A., T. Brinkhoff, = G. Muyzer, D. P. Moser, J. Rethmeier, H. W. Jannasch, 2000. Appl. Environ. Microbiol., 66, 3125-3133.

15. Waksman, S. A, 1922. J. Bacteriol., 7<= /b>, 605-608.

16. Waksman, S. A., J. S. Jof= fe, 1922. J. Bacteriol., 7, 239-256.

ИЗОЛИРА= ;НЕ И ХАРАКТЕРИЗh= 8;РАНЕ НА СЯРА-ОКИС= 051;ЯВАЩИ БАКТЕРИИ<= /b>

Раджагопа= 083; Видиалакшмl= 0;¹*, Р. Сридар²

Ре = 79;юме

Изl= 6;лирани са сяра-окисля = 74;ащи бактерии от = 088;азлични проби – ризосфера н = 72; оризище и бо= 073;ови култури, канализациl= 6;нни води, биогаз= 085;а тиня, отпадн= 080; води при обработка н = 72; кожи и минна почва. От получените 28 изолата 14 са скринирани на база на ефикасностm= 0;а им да понижават р = 53; на растежната среда от 8.0 до £5.0. Избраните изолати са характеризl= 0;рани и отнесени към род Thiobacillus.=

&= nbsp;

Name of SOB isolate	pH Reduction in growth media
Starkey broth	NCL broth	Thiosulphate broth
SR	4.5	8.0	6.5
SP2	4.5	8.0	5.0
SN	6.5	8.0	8.0
SS	5.5	5.5	8.0
NP2	5.0	5.0	5.0
NS	8.0	5.5	6.0
TP2	4.5	5.5	4.0
TB	5.5	5.5	8.0
TR	8.0	8.0	6.0
ASE	8.0	6.0	4.5
ATE	8.0	6.5	8.0
ATR	8.0	6.0	5.5
ASS	6.0	5.5	6.0
ATB	6.0	6.0	8.0

Isolates	Morphology	Gram reaction	Utilization	Influence of biotin	Colony character	Nutritional type
Elemental sulphur	Thio-sulphate	Glucose
SR	Short rods	Negative	+	+	+	+	Smooth, round straw yellow colour colonies	Hetero- troph
SP2	Short rods	Negative	+	+	+	+
SN	Short rods	Negative	+	-	+	+
SS	Short rods	Negative	+	-	+	+
NP2	Short rods	Negative	+	+	+	+
NS	Short rods	Negative	+	+	+	+
TP2	Short rods	Negative	+	+	+	+
TB	Short rods	Negative	+	+	+	+
TR	Short rods	Negative	-	+	+	+
ASE	Short rods	Negative	+	+	-	+	Smooth, raised pink colour colonies	Auto- troph
ATE	Short rods	Negative	-	+	-	+
ATR	Short rods	Negative	-	+	-	+
ASS	Short rods	Negative	+	+	-	+
ATB	Short rods	Negative	+	+	-	+

Summary

Growth of type culture. The strain Th= iobacillus thiooxidans MTCC 468 was obtained from the Microbial Type Culture Collection (MTCC), Chandigarh= , Haryana and was grown in the medium as recommended by the MTCC and used as a reference culture.

Isolation and screen= ing of sulphur oxidizing bacteria

Fig.1. Reduction of pH in the growth media by sulphur oxidizing bacteria.<= o:p>

= ;

Characterization of = the selected isolates

Discussion

References