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Isolation and characterization of facultatively autotrophic, acidophilic, thermophilic bacterium, Thiobacillus thermosulfatus sp. Nov. from coal mine overburden spoil.


Coal mine overburden spoil presents a physically disturbed habitat with almost all vital nutrients including organic carbon, nitrogen and phosphorous deficient situation. Since, mine overburden represents hostile environment but rich is sulfur and iron components in the form of pyrite, the expected group of bacteria in fresh mine overburden spoil are chemolithotrophic, sulfur and iron oxidizers. Auto-oxidation of pyrite consequently leading to the burning of coal and rise of temperature in mine spoil. Hydrogen sulfide, which is one of the major out comes gets converted into sulfuric acid causing a decline in pH and hence resulting an acidic environment (Kristjansson and Stetter, 1992; Kristjansson et al., 2000). Thus, thermo-acidic environmental condition in fresh coal mine spoil constrains the successional/developmental process of the microorganisms as well as impedes the spoil reclamation process.

Sulfur oxidizers are taxonomically heterogeneous group of bacteria, grow near the source of sulfur (i.e. sulfur mines, hot sulfur springs, coal mine spoil) capable of oxidizing sulphides, elemental sulfur, sulphite and ultimately converted them to sulphate. Similarly, iron oxidizers found growing at the sites of geological deposits of iron sulphide minerals (pyrite: FeS2 and chalcopyrite: CuFeS2) oxidized the ferrous to ferric forms and utilized the energy released there upon and hence are chemolithoautotrophic in nature. The ability of sulfur and iron oxidizing bacteria to convert sulphide ores to water soluble heavy metal sulphates, is utilized for leaching of low grade ores.

Size of microbial pool is considered to be a functional index of soil (Carter, 1986; McGill et al., 1986, Smith and Paul, 1988) and is considered as an ecological marker of soil health (Smith and Paul, 1990) because of their involvement in microbial cycling of carbon, nitrogen and phosphorus (Nannipieri et al., 1990; Beyer et al., 1992; Duxbury and Nkambule, 1994; Filefibach et al., 1994; Dick et al., 1996). Thus, the isolation and characterization of microorganisms provide insight into the microbial biotransformation process (Nannipieri et al., 1980; Skujins and Richardson, 1985; Riffaldi et al., 2002). Several authors have attempted the bacteriological study in terms of lower microbial abundance and activity in fresh mine spoil (Norris, 1990; Tyson et al., 2005). Presence of acidophilic bacteria in the coal mine spoil have been documented by several workers (Wood and Kelly, 1986; Norris, 1990; Kristjansson and Stetter, 1992; Shennan, 1996; Rawlings et al., 1999; Kristjansson et al., 2000; Tyson et al., 2004). As per Baker and Banfield (2003), Proteobacteria, Nitrospira, Firmicutes and Acidobacteria dominate the microbial populations in coal mining site. This species is metabolically flexible and uses ferrous iron and reduced inorganic sulfur compounds as an electron donor, to produce ferric iron and sulfuric acid (Prescott et al., 1999). The review thus indicated either low bacterial abundance or the presence of specific group of bacteria that have physiological adaptive strategy for thermo-acidic tolerance in the coal mine overburden spoil.

The present study was designed to isolate chemolithotrophic bacteria from fresh coal mine overburden spoil and to study the growth pattern with respect to different nutrient supplements (i.e. tetrathionate, elemental sulfur, yeast extract, glutamate, succinate and thiocyanate) in the culture medium followed by their thermal and acid tolerance ability. Further, in order to determine the presence of contaminating heterotrophic bacteria, growth was monitored by determining viable cell counts on both solid medium-A and medium-H.

Materials & Methods

Study site

The present study was carried out in the Basundhara (west) open cast colliery, Ib valley coalfields area of Mahanadi Coalfields Limited (MCL), Sundargarh, Orissa, India (Geographical location: between 22[degrees] 03' 58"-20[degrees] 04' 11" north latitude and 83[degrees] 42' 46"-83[degrees] 44' 45" east longitude). The study area experiences a dry, hot and semiarid climate with annual rain fall of 1514 mm [yr.sup.-1], annual average temperature of 26[degrees]C and relative humidity of 15%. Open cast coal mining activities in the study site lead to the formation of mine spoil overburden.


Sampling was done in accordance with the general methods for soil microbiological study (Parkinson et al., 1971). Coal mine spoil samples were collected from the fresh mine overburden following aseptic procedure. Each site was divided into 3 blocks, and during each sampling five soil samples were collected randomly from 0-15cm soil depth by digging pits of (15 x 15 x 15)[cm.sup.3] size in each block. Five sub-samples collected from each block were thoroughly mixed to form one composite sample, homogenized and subjected to sieving (0.2mm mesh) and stored in pre-sterilized screw capped Falcon tubes at 4[degrees]C until analyzed.

Isolation of bacteria

Isolation of Thiobacillus thermosulfatus was done by serial dilution technique following the procedure of Shooner et al. (1996). Spoil sample (~1g) was amended with 0.5% (w/v) tyndallized [S.sup.0] powder in a 250ml Erlenmeyer flask and incubated at 50[degrees]C in a rotatory shaker. Aliquot of 100ul from the flask was transferred to medium-A [K[H.sub.2]P[O.sub.4]-2.04g, [(N[H.sub.4]).sup.2]S[O.sub.4]-0.2g, Ca[Cl.sub.2][H.sub.2]O-0.2g, MgS[O.sub.4.]7[H.sub.2]O-0.5g, bromophenol blue- 0.02g per liter and pH adjusted to 6.0 with 1N [H.sub.2]S[O.sub.4] and then [S.sup.0] powder (0.5% w/v) was added]. After the growth of the bacteria in medium-A, the bacterial cells were transferred to a medium whose composition is same as that of medium-A except the [S.sup.0] powder, which was substituted with 20mM sodium thiosulfate (pH- 6.0, adjusted with 1N [H.sub.2]S[O.sub.4]). After a few transfer, [S.sup.0] was replaced by 20mM sodium thiosulfate. The inoculated bacteria were subjected to incubation at 50[degrees]C for 20hr. After this, 100ul of serially diluted (10-5 fold) bacterial suspension was transferred onto a sterilized petridish containing 20mM sodium thiosulfate and 0.6% (w/v) gelrite (Lin and Casida, 1984) and subjected to incubation at 50[degrees]C for 5 days. Presence of heterotrophic-sporulated bacteria if any, was checked through out the isolation process by inoculating the bacterial inoculum into medium-H [Glucose- 1g, Tryptone- 5g, Yeast extract-2.5g, Ca[Cl.sub.2][H.sub.2]O-0.57g and Gelrite (0.6%)- 6g per liter]. Absence of the contaminating bacteria growth in medium-H confirms the absence of heterotrophic sporulating bacteria and therefore such observation confirms the isolation of Thiobacillus thermosulfatus in pure form. Isolated bacterium from the culture suspension was studied further microscopically for their shape and Gram's stain response.

Gram's staining

The microbial sample was smeared on a sterilized glass slide and heat fixed. One or two drops of crystal violet solution were added to the smear followed by gram's iodine. After few minutes, the slide was washed with alcohol, dried and counterstain with safranine. The slide was then washed with water, dried and observed under the microscope.

Bacterial growth pattern and pH analysis

Bacterial growth experiment in case of Thiobacillus thermosulfatus was performed at 50[degrees]C by using Medium-A supplemented with thiosulfate (20mM). Bacterial growth was monitored by measuring the absorbance at 400nm at different time intervals upto 40hr of incubation. Simultaneously, the change in pH of the culture medium due to the bacterial growth was also recorded. In addition, experiments were also performed to determine the optimal pH and temperature required for maximal growth of the bacterium by using medium-A supplemented with 20mM thiosulfate. The pH was controlled automatically and growth was monitored by measuring the absorbance at 400nm as well as bacterial counts at different time intervals. Further, growth of Thiobacillus thermosulfatus was also tested with respect to different substrates [i.e. tetrathionate (10mM), elemental sulfur (0.5%), yeast extract (0.05%), glutamate (10mM), succinate (10mM) and thiocyanate (10mM)] supplemented in the culture medium. Specific growth rate was calculated from the expression: k = ln2/([t.sub.2]-[t.sub.1]), where ([t.sub.2]-[t.sub.1]) was the time interval required for the bacterial count to double during the exponential growth phase.

Determination of thermal death time (TDT)

Medium-A supplemented with thiosulfate (20mM) inoculated individually with 100ul of Thiobacillus thermosulfatus culture were subjected to heat treatment at 80[degrees]C for different time intervals (30min, 45min, 1hr, 75min, 90min and 2hr) and the heat-treated inoculums were streaked individually on different petridishes with presterilized solid medium-A with 20mM thiosulfate. The plates were incubated at 37[degrees]C for 24hr for the development of colonies.


Isolation of Thiobacillus thermosulfatus resulted discrete colonies on medium-A, which were observed microscopically to be the mixture of Thiobacillus thermosulfatus and some gram positive spore forming bacillus species. After subsequent sub-culturing on medium-A with gelling agent such as agar, gerlite, the presence of spore-formers was further checked on medium-H. No growth of spore-formers was observed after incubation for 5 days at 50[degrees]C, which confirmed the elimination of heterotrophic bacillus contaminants from medium-A and hence confirms the isolation of Thiobacillus thermosulfatus in pure form. The bacterial colonies isolated from solid medium-A were found to be small (< 1mm), translucent and having sulfur deposits in the center, gram negative, rod shaped, facultatively autotrophic, strictly aerobic colorless sulfur bacterium (Figure 1a & b).



Growth experiments were performed under chemolithotrophic culture condition at 50[degrees]C using medium-A supplemented with thiosulfate (20mM). The growth curve of Thiobacillus thermosulfatus revealed that the lag phase continued upto 4hr of incubation. The exponential phase started thereafter till 15hr of incubation. It was further observed that the bacterial growth was arrested between 15hr to 20hr of incubation. The growth of the bacterium again continued upto 35hr of incubation, after which the bacteria entered into the stationary phase (Figure 2). Specific growth rate was calculated to be 0.23[6hr.sup.-1]. As revealed from the experimental data, the pH of the culture medium slowly increased from 6.0 to 7.5 with the increase in biomass up to 17hr of incubation. However, at the end of the growth, the pH of culture medium showed a decline trend and dropped down to 2.1 at 40hr of incubation. During the pH controlled experiments, the maximum growth rate was obtained at pH values between 5.2 (0.230[hr.sup.-1]) and 5.6 (0.236[hr.sup.-1]) and temperature between 50[degrees]C (0.236[hr.sup.-1]) and 53[degrees]C (0.224[hr.sup.-1]).


Further, growth analysis of Thiobacillus thermosulfatus in medium-A with different nutrient supplements suggested that profuse bacterial growth was exhibited in medium-A supplemented with thiosulfate, elemental sulfur, yeast extract, minimal growth with glutamate and succinate, where as bacterial growth did not occur when supplemented with thiocyanate (Table 1). Heterotrophic growth occurred on media containing yeast extract, glutamate and succinate, which was observed by the turbidity and plate count methods. Characteristic odor was also noticed at the end of growth on organic and inorganic substrates. However, under anaerobic conditions, no growth was occurred under autotrophic and heterotrophic conditions.

Thiobacillus thermosulfatus showed moderate growth even exposed at 80[degrees]C for 15min. Thermal death time of Thiobacillus thermosulfatus in chemolithotrophic culture conditions was found to be 1hr at 80[degrees]C (Table 2). Thermal death time analysis revealed that an increasing trend of death of bacterial strain was observed with the increase in exposure time at 80[degrees]C for chemolithotrophic culture condition.


Coal mine overburden spoil is a unique habitat with extreme environment. Because of the pyrite auto-oxidation, fresh coal mine spoil is associated with high temperature, acid production and devoid of available organic nutrients, and expected to be inhabited by the extremophiles such as thermo-tolerant heterotrophs (Norris et al., 1986; Kuenen et al., 1992; Shennan, 1996; Rawlings et al., 1999 and Akbar et al., 2005), acid tolerant bacteria (Wood and Kelly, 1986; Norris, 1990; Kristjansson and Stetter, 1992; Shennan, 1996; Wulf-Durand et al., 1997; Rawlings et al., 1999; Kristjansson et al., 2000; Akbar et al., 2005). When the fresh mine spoil was processed for heterotrophic isolation with medium-H, it was observed to be inhabited by relatively much low heterotrophic bacterial isolates as compared to chemolithotrophs (Harrison, 1984; Wood and Kelly, 1986; Kuenen et al., 1992; Kristjansson and Stetter, 1992; Madigan et al., 1997; Kristjansson et al., 2000; Boone et al., 2001; Fujiwara, 2002).

Following aseptic protocol and media, it could be possible for isolation, cultivation and maintenance of Thiobacillus thermosulfatus (ther. mo. sul. fa' tus. Gr. n. thermos, heat; L. n. sulfatus, sulfur; L. adj. thermosufatus) culture using medium-A enriched with elemental sulfur, which does not support the development of other autotrophs (Temple and Colmer, 1951; Tuovinen and Kelly, 1974). These organisms have the ability to derive energy by the oxidation of inorganic sulfur compounds [[Na.sub.2][S.sub.2][O.sub.3], [(N[H.sub.4]).sub.2]S[O.sub.4], MgS[O.sub.4]] as well as [S.sub.0] powder under acidic condition, which is the distinct key character employed in the isolation procedure. In addition, thiosulfate is the commonly used substrate for the growth of sulfur oxidizers, as it is easily soluble and reasonably stable at the pH range suitable for Thiobacillus thermosulfatus. The bacterial culture triggered the chemical degradation of [Na.sub.2][S.sub.2][O.sub.3] present in the medium, leads to the formation of (colloidal) sulfur in acidic media depending on the ionic composition (Shooner et al., 1996; Atlas and Bartha, 1998; Rawlings et al., 1999; Kelly and Wood, 2000).

The bacterium showed diauxic growth pattern in medium-A due to the availability of two nutrients, such as thiosulfate first and later tetrathionate, as an intermediate in the culture medium (Tuovinen and Kelly, 1974; Wood and Kelly, 1986; Shooner et al., 1996). Growth analysis of Thiobacillus thermosulfatus indicated that the bacterium exhibited a specific growth rate of 0.236[hr.sup.1]. Simultaneously, the corresponding change in pH showed a decline trend (pH-6.0 to 2.1) due to acid production during bacterial growth at 50[degrees]C. Further, the pH controlled experiments suggested that optimum growth of Thiobacillus thermosulfatus was achieved at pH 5.2 to 5.6 and temperature 50[degrees]C to 53[degrees]C (Shooner et al., 1996).

Thermal response of the isolated bacteria suggested that Thiobacillus thermosulfatus grow chemolithotrophically at 80[degrees]C for 45min and thus, thermal death time of Thiobacillus thermosulfatus was found to be 1hr at 80[degrees]C. Since, Thiobacillus thermosulfatus showed optimum growth in medium-A supplemented with sulfur derivatives at 50[degrees]C and pH-5.6, the bacterium is concluded to be a chemolithotrophic, facultatively autotrophic, acidophilic, moderately thermophilic bacterium.


Coal mine spoil overburden represents a disequllibriated geomorphic system due to the altered physical and chemical structure, which is supported by some bacterial community with specialized physiological adaptability. Thiobacillus thermosulfatus isolated from the fresh coal mine overburden spoil was ascertained to be gram negative, rod shaped, motile and chemolithotrophic strain. Optimum growth of Thiobacillus thermosulfatus occurs at pH 5.2 to 5.6 and temperature 50[degrees]C to 53[degrees]C suggesting that the bacterium is acidophilic and moderately thermophilic in nature. Further, growth study revealed that the bacterium is strictly aerobic and facultatively autotrophic i.e. grows autotropically on thiosulfate, elemental sulfur and tetrathionate, and heterotropically on yeast extract, succinate and glutamate. During elemental sulfur oxidation, a considerable amount of sulphate is produced. From an ecological point of view, microbiological investigation involving exploration and physiological profiling of bacteria would result isolation of unique bacterial strains having bioprospecting potential, such as bioproduction of sulfate at 50[degrees]C in presence of elemental sulfur was observed not only in mine overburden spoil samples but also in sludge samples obtained from municipal waste water treatment plants and paper/pulp industries.


Author is thankful to Prof. Niranjan Behera, School of Life Sciences, Sambalpur University, Orissa for providing laboratory facilities and critical suggestions. Author also likes to thank Ms. Jyoti Patel and Ms. Rumi Chakraborty for timely help and assistance.


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Amiya Kumar Patel

Division of Biotechnology, Majhighariani Institute of Technology and Science (MITS), At--Sriram Vihar, Bhujbala, Po-Kolnara, Rayagada, (Pin- 765017), Orissa, India E-mail:
Table 1: Growth of Thiobacillus thermosulfatus in Medium-A
with different ingredients. (+++: profuse growth; ++: moderate
growth and +: minimal growth).

Ingredients Bacterial growth pattern

Thiosulfate +++
Tetrathionate ++
Elemental sulfur ++
Yeast extract ++
Glutamate +
Succinate +
Thiocyanate No growth

Table 2: Thermal response of Thiobacillus thermosulfatus at
80[degrees]C under chemolithotrophic culture condition.
(++ : moderate growth and + : minimal growth).

Exposure time (min) Thermal response at 80[degrees]C

15 min ++
30 min +
45 min +
1 hr No growth
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Publication:International Journal of Applied Environmental Sciences
Date:Feb 1, 2010
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