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Isolation and Characterization of Thiobacillus Ferrooxidans from Coal Acid Mine Drainage

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Isolation and Characterization of Thiobacillus Ferrooxidans from Coal Acid Mine Drainage International Journal of Applied Agricultural Research ISSN 0973-2683 Volume 5 Number 1 (2010) pp. 73–85 © Research India Publications http://www.ripublication.com/ijaar.htm Isolation and Characterization of Thiobacillus ferrooxidans from Coal Acid Mine Drainage 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: [email protected] Abstract Coal mine drainage refers to the acidic drainage caused by surface mining, deep mining or coal refuse piles. Being highly acidic with elevated levels of dissolved metals, it is otherwise known as “Acid Mine Drainage (AMD)”. Formation of AMD is due to a series of geochemical and microbiological processes involving acidophilic chemolithotrophs such as Thiobacillus, Thiomispora, Leptospirillum etc. Presence and growth of these bacteria in coal mine drainage further decreases pH, making the drainage more acidic. Such highly acidic drainage when mixes with other water bodies disrupt the normal aquatic food web. In the present study, a chemolithotrophic, acidophilic, iron- oxidizing, gram-negative, stricked shaped bacteria i.e Thiobacillus ferrooxidans preferring temperature range 20-35°C was isolated and microbiological characterization was performed. The study revealed that it is an aerobic, mesophilic, acidophilic bacteria having variable pH tolerance range (2-8). Detailed growth analysis revealed its chemoautotrophic, mixotrophic and heterotrophic mode of growth. Heterotrophic and mixotrophic growth of bacteria with added carbon substrate led to the improvement of pH of the culture medium indicating the cessation of chemolithotrophic activity. The study therefore suggested that supplementation of coal mine drainage with organic carbonaceous substrate can be one of the effective environmental management strategy for minimizing acid production by the chemolithotrophs. Key words: Coal mine drainage, AMD, chemolithotrophs, Thiobacillus, bacterial growth. 74 Amiya Kumar Patel Introduction Acid mine drainage (AMD) is formed by a series of complex geochemical and microbial reactions, which is primarily a function of geology, hydrology and physico- chemical properties of mine spoil caused by surface mining, deep mining or coal refuse piles. When water comes in contact with coal mine overburden with elevated concentration of dissolved sulfates, ferric iron and other heavy metals and extremely low pH, the development of iron-oxidizing bacteria is favored in AMD (Johnson and Rang, 1993; Schleper et al., 1995; Hallberg and Johnson, 2001, 2003), which can seriously degrade the aquatic habitat because of toxicity, corrosion, incrustation etc. However, the major source of acidity is due to the oxidation of pyrite (FeS2) that is 2+ 2- + exposed by coal mining to release dissolved Fe , SO4 and H , followed by further oxidation of Fe2+ to Fe3+ and the precipitation of the iron as a hydroxide producing more H+ (Atlas and Bartha, 2005). AMD occurs due to a series of microbiological oxidation processes involving acidophilic chemolithotrophs and low pH values speed up the acid-forming reaction (Cravotta et al., 1994), which can be explained in the form of following equations: 2- + FeS2(s) + 3.75 O2 + 3.5 H2O = Fe(OH)3(s) + 2 SO4 + 4 H + heat (1.1) 2+ 2- + FeS2(s) + 3.5 O2 + H2O = Fe + 2 SO4 + 2H (1.2) 2+ + 3+ Fe + 0.25 O2 + H = Fe + 0.5 H2O (1.3) 3+ 2+ 2- + FeS2(s) + 14 Fe + 8 H2O = 15 Fe + 2 SO4 + 16 H (1.4) 3+ + Fe + 3 H2O = Fe(OH)3(s) + 3 H (1.5) Many factors determine the rate of AMD generation from pyrite oxidation including the activity of bacteria (Wakao et al., 1988; Ehrlich, 1990; Chavarie et al., 1993; Rawlings et al., 1999; Baker and Banfield, 2003; Hallberg & Johnson, 2003), pH (Kleinmann et al., 1981; Nordstrom, 1982; Harrison, 1985; Sand, 1989; Amaro et al., 1991; Hallberg and Johnson, 2001), pyrite chemistry and surface area (McKibben and Barnes, 1986; Ferguson and Erickson, 1988; Rawlings et al., 1999), temperature (Evans and Rose, 1995; Schleper et al., 1995; Rawlings, 1999; Bond et al, 2000) and O2 concentration (Watzlaf, 1992). Further studies have also indicated that AMD generation due to the chemolithotrophic oxidation, which is much faster than the geochemical oxidation (Nordstrom, 1982, Hornberger et al., 1990; Moses and Herman, 1991; Alpers et al., 1994; Williamson and Rimstidt, 1994; Evangelou, 1995; Nordstrom and Alpers, 1996; Rawlings et al., 1999; Kelly and Wood, 2000). The bacteria present in AMD mostly belong to genus: Thiobacillus and Thiomispora. Among the chemolithotrophs, members of the genus Thiobacillus are the prominent bacteria, which oxidize ferrous iron (Temple and Colmer, 1951; Kelly and Wood, 2000) and inorganic sulfur compounds including pyrite like metal sulfides (Touvinen and Kelly, 1974; Ehrlich, 1990) to derive energy for their autotrophic growth. Further, microbiological studies revealed that both sulfur- and iron-oxidizing bacteria such as Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans were present in AMD (Moreira and Amils, 1997; Friese et al., 1998; Hairashi et al., 1998; Kelly and Wood, 2000; Lo´pez-Archilla and Amils, 2001). Thiobacillus ferrooxidans, Leptospirillum sp. and Ferroplasma sp. have also been considered principally responsible for the extreme conditions of AMD (Sand et al., 1992; Schrenk et al., 1998; Edwards et al., 1999, 2000). Besides these, the physiological Isolation and Characterization of Thiobacillus 75 polymorphism of these bacteria have been noted and explained by several workers (Kuenen et al., 1991; Muyzer and Uitterlinden, 1993; Leduc and Ferroni, 1994; Chisholm et al., 1998; Rawlings, 1999; Frattini et al., 2000; Ageeva et al., 2001; Ito et al., 2002). Keeping this concept into view, the objective of the investigation was to isolate a chemolithotrophic bacterial strain i.e. Thiobacillus ferrooxidans from the coal mine AMD and studied its microbiological characteristics in terms of Gram stain response, bacterial growth pattern subjected to change in pH in chemolithotrohic, mixotrophic and heterotrophic culture conditions, thermal death time determination and antibiotics sensitivity, with an aim to mitigate the problem of acidity of the coal mine drainage. Materials & Methods Study site The coal mine AMD samples were collected from Basundhara (west) colliery, Mahanadi Coalfields Limited, Gopalpur region of Sundargarh district, Orissa (India). The study site is subjected to open caste mining since 1990. One of the major impurities of coal is pyrite (FeS2) and this being exposed to atmosphere after mining results in acid mine drainage come out of the mining pits and piles. The discharge at its origin maintains a pH of 2.5 and subsequently the pH was found to be 4.5. The discharge after flowing some distance become yellowish in colour due to the precipitates of Fe(OH)3. Sampling Samples of coal mine AMD were collected (n = 10) at the point of origin from the above mentioned coal mine area and mixed together to form a composite sample following aseptic procedure. For this, pre-sterilized screw capped Falcon tubes of 15ml capacity were used to collect AMD samples. Collected discharge samples were immediately subjected to fixation with 3% paraformaldehyde in phosphate buffered saline (PBS) solution [pH 7.4 at 25°C]. The initial pH of the coal mine drainage was measured at the spot using Handy pH meter (Elico) and recorded. Isolation of bacteria About 100µl of coal mine drainage sample was inoculated in 50ml of modified ferrous sulfate medium [Na2S2O3- 10g, (NH4)2SO4- 0.3g, Yeast extract- 5g, FeSO4.7H2O- 10g, K2HPO4- 4g, KH2PO4- 1.5g, MgSO4- 0.5g per liter with the initial pH adjusted to 4.0 with 1N H2SO4] (Temple and Colmer, 1951; Tuovinen and Kelly, 1974) and incubated at 35°C for 48hr. Isolation of Thiobacillus ferrooxidans was performed by serial dilution technique followed by streaking 100µl of AMD sample onto pre-solidified ferrous sulfate agar medium. Isolated bacterium from the culture suspension was studied further microscopically for their shape and Gram’s stain response. 76 Amiya Kumar Patel 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 The isolated bacterium from the ferrous sulfate medium was further processed for growth analysis of Thiobacillus ferrooxidans with three different combinations of nutrients in chemolithotrophic (Na2S2O3 & Yeast Extract), mixotrophic (Glucose & Na2S2O3) and heterotrophic (Glucose & Yeast Extract) culture conditions. To 25ml of ferrous sulfate medium (i.e. chemolithotrophic, mixotrophic and heterotrophic culture composition) taken in each conical flask, 100µl of Thiobacillus culture from chemolithotrophic master culture was inoculated and subjected to incubation at 35ºC. The culture flasks were subjected to rotatory shaking at 180rpm and the growth curve analysis was done by taking absorbance at 640nm at different time intervals starting from control till 30hr. Simultaneously, the change in pH of the culture was also recorded. Specific growth rate (µ) was also calculated for each culture condition of Thiobacillus ferrooxidans. Specific growth rate (µ) was calculated as follows: log N – log N
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