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 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 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 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 µ 10 t 10 0 = tt – t0 2.303

Where, N0 = Absorbance at the initiation of the exponential phase of growth. Nt = Absorbance at the mid/end of the exponential phase of growth. (tt –t0) = time difference to achieve absorbance from N0 to Nt.

Determination of thermal death time (TDT) Ferrous sulfate medium inoculated individually with 100µl of three different Thiobacillus culture (chemolithotrophic, mixotrophic and heterotrophic) were subjected to heat treatment at 60ºC for different time intervals (15min, 30min, 45min, 1hr, 75min, 1½hr, 2hr, 2½hr, 3hr) and the heat-treated inoculums were streaked individually on different petridishes with the respective ferrous sulfate agar medium composition as mentioned earlier for chemolithophic, mixotrophic and heterotrophic culture. The plates were kept at 37ºC for 24hr in an incubator for the development of colonies.

Antimicrobial activity Suspension of 100µl of different antibiotics such as ampicillin, amoxycillin, benzylpenicillin, chloramphenicol, ciprofloxacin, erythromycin, kanamycin, gentamicin, ofloxacin, rifampicin, streptomycin, tetracycline, tobramycin having the same concentration (0.5mg/ml of distilled water) were introduced into the wells prepared in the petridishes containing ferrous sulfate agar medium streaked with Thiobacillus ferrooxidans culture. The petridishes were incubated at 37ºC for 24hr to visualize the zone of inhibition. Similar set up of experiments were performed for Isolation and Characterization of Thiobacillus 77 chemolithotrophic, mixotrophic and heterotrophic culture conditions. By measuring the diameter of zone of inhibition (in mm) around the wells, the degree of sensitivity of Thiobacillus ferrooxidans to different antibiotics under different culture conditions was ascertained.

Results Bacterial colonies of Thiobacillus ferrooxidans appeared on ferrous sulfate agar after incubation at 35ºC for 48hr were observed to be smooth, circular, low convex and greater opacity of their size (Figure 1A). The diameter of the colonies were found to be approximately (1-2) mm. During the development of colony, there was a change in colour of the medium from yellow to green. The bacterial cells present in the coal mine drainage, when microscopically observed were found to be rod shaped and Gram negative (Figure 1B). Under chemolithotrophic (Na2S2O3 & Yeast Extract) cultural condition, lag phase of Thiobacillus ferrooxidans continued upto 3hr of incubation. The log phase started thereafter till the 26hr of incubation after which stationary phase started. As revealed from the experimental data, initially pH of the culture medium before the bacterial growth was maintained at 4.0. However, at the end of the growth at 30hr of incubation, pH value showed a decline trend and it dropped down to 1.95 (Figure 2A). However, the heterotrophic (Glucose & Yeast Extract) growth condition showed that the lag phase of bacterium continued upto 3hr of incubation followed by log phase upto 20hr. The pH due to bacterial growth in heterotrophic culture medium increased from 4.0 to 6.17 at 30hr of incubation (Figure 2B). Similarly, in case of mixotrophic (Glucose & Na2S2O3) growth condition, the lag phase was continued upto 7hr and was followed by log phase upto 20hr of incubation. The stationary phase was then initiated followed by change in pH value from 4.0 to 5.1 at 30hr of incubation (Figure 2C). Thermal death time of Thiobacillus ferrooxidans in chemolithotrophic, mixotrophic and heterotrophic culture conditions was found to be 75min, 60min and 45min respectively at 60ºC. Thermal death time analysis of Thiobacillus ferrooxidans revealed that an increasing trend of death of bacterial strain was observed with the increase in exposure time at 60ºC for all the three culture conditions (i.e. chemolithotrophic, mixotrophic and heterotrophic). The antibiotic sensitivity test indicated that Thiobacillus ferrooxidans grown under chemolithotrophic, mixotrophic and heterotrophic culture condition showed clear differential zone of inhibition towards the antibiotics such as chloramphenicol, ciprofloxacin, gentamycin, ofloxacin, rifampicin, streptomycin, tetracycline and tobramycin. However, the bacterium did not show any sensitivity to some antibiotics such as ampicillin, amoxycillin, benzylpenicillin, erythromycin and kanamycin (Ampres Amores Benres Chlsen Cipsen Eryres Kanres Gensen Oflsen Rifsen Strsen Tetsen Tobsen) (Table 1).

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Figure 1(A): Chemolithotrophic culture of Figure 1(B): Gram staining showing Thiobacillus ferrooxidans showing colonies. Gram- negative, rod shaped Thiobacillus ferrooxidans.

Figure 2: Growth curve of Thiobacillus ferrooxidans in culture medium supplemented with (A) Na2S2O3 & yeast extract (chemolithotrophic growth); (B) glucose & yeast extract (heterotrophic growth); (C) glucose & Na2S2O3 (mixotrophic growth). Symbols: ■→ chemolithotrophic/ heterotrophic/ mixotrophic mode of growth;  → pH.

Isolation and Characterization of Thiobacillus 79

Table 1: Effect of different antibiotics on Thiobacillus ferrooxidans having concentration (0.5mg/ml) in chemolithotrophic (Na2S2O3 & yeast extract), mixotrophic (glucose & Na2S2O3) and heterotrophic (glucose & yeast extract) culture conditions. The diameter of zone of inhibition (mean of 5 replicates) is expressed in (mm ± SD).

Diameter of zone of inhibition (mm) in Name of Thiobacillus ferrooxidans culture Antibiotics CHEMOLITHOTROPHIC MIXOTROPHIC HETEROTROPHIC (Na2S2O3 + Yeast Extract) (Glucose + Na2S2O3) (Glucose + Yeast Extract) Ampicillin ------Amoxycillin ------Benzylpenicillin ------Chloramphenicol 20 ± 2 28 ± 1 26 ± 3 Ciprofloxacin 31 ± 1 25 ± 2 34 ± 2 Erythromycin ------Kanamycin ------Gentamycin 24 ± 1 30 ± 3 27 ± 1 Ofloxacin 30 ± 2 26 ± 2 32 ± 3 Rifampicin 30 ± 1 22 ± 1 34 ± 2 Streptomycin 14 ± 2 18 ± 2 16 ± 1 Tetracycline 18 ± 3 28 ± 2 30 ± 3 Tobramycin 24 ± 1 36 ± 1 26 ± 1

Discussion Ferrous sulfate medium was used for the isolation, cultivation and maintenance of Thiobacillus ferrooxidans. These organisms have the ability to derive energy by the oxidation of inorganic sulfur compounds [Na2S2O3, (NH4)2SO4, MgSO4] as well as ferrous iron (FeSO4.7H2O) under acidic condition, which is the distinct key character employed in the isolation procedure. In addition, thiosulfate is the commonly used substrate for growth of the sulfur oxidizers, as it is easily soluble and reasonably stable at the pH range suitable for Thiobacillus ferrooxidans. Pure culture of Thiobacillus ferrooxidans can be easily obtained on the ferrous iron containing medium that do not support the development of other autotrophs (Temple and Colmer, 1951; Tuovinen and Kelly, 1974). The bacterial culture triggered the chemical degradation of Na2S2O3 present in the medium, leads to the formation of (colloidal) sulfur in acidic media depending on the ionic composition (Rawlings et al., 1999; Kelly and Wood, 2000; Atlas and Bartha, 2005) and hence change its colour from yellow to green, which suggested that the bacterium is acidophilic, obligately and facultatively chemolithotrophic strain (Sand, 1989; Cravotta et al., 1994; Rawlings et al., 1999; Hallberg and Johnson, 2001; Edward and Banfield, 2002; Baker and Banfield, 2003). 80 Amiya Kumar Patel

Microscopic studies revealed that the majority of the bacterial population present in the coal mine drainage in acidic pH condition (pH- 4.0) was found to be Gram negative (Bacelar-Nicolau and Johnson, 1999; Brofft et al., 2002; Hallberg and Johnson, 2001), which appears pink in colour and rod shaped structure. Specific growth rate (µ) in case of chemolithortrophic culture condition was calculated to be 0.101 hr-1, which showed close resemblance with the findings by Eccleston and Kelly (1978). The data revealed that the growth of Thiobacillus ferrooxidans was slow and sustained for a longer period in the chemolithotrophic culture condition due to low energy yielding states (Atlas and Bartha, 2005). Therefore, yeast extract was taken as growth factor and source of biotin, which accelerates the growth of the bacteria. Specific growth rate (µ) in case of heterotrophic culture condition was calculated to be comparatively less (0.063 hr-1) as compared to the chemolithotrophic culture condition, which might be due to the switching over of the bacterium from chemolithotrophic to heterotrophic condition. However, specific growth rate (µ) in case of mixotrophic culture condition was calculated to be 0.137 hr-1 i.e. greater than the chemolithotrohic and heterotrophic conditions suggesting that Thiobacillus ferrooxidans has the capacity to derive energy both from inorganic as well as organic nutrients supplemented in the mixotrophic culture condition. Thus, the study indicated that the chemolithotrophic bacteria have a versatile physiology and are observed to switch over to heterotrophic (Glucose & Na2S2O3) and mixotrophic (Glucose & Na2S2O3) culture conditions (Matin and Rittenberg, 1971; Bond and Banfield, 2000; Hallberg and Johnson, 2003). Further, the change in pH due to the bacterial growth in chemolithotrophic culture condition form 4.0 to 1.95 over 30hr of incubation suggested Thiobacillus ferrooxidans as an acidophilic bacteria (Cravotta et al., 1994; Bacelar-Nicolau and Johnson, 1999; Rawlings et al., 1999; Edwards et al., 2000; Baker and Banfield, 2003; Hallberg and Johnson, 2003). Comparative study on thermal death time (TDT) of Thiobacillus ferrooxidans in chemolithotrophic, mixotrophic and heterotrophic culture conditions suggested that the bacterium under chemolithotrophic culture condition was found to be more thermo-tolerant as compared to mixotrophic and heterotrophic culture conditions (Evans and Rose, 1995; Schleper et al., 1995; Rawlings, 1999; Bond et al, 2000; Hallberg and Johnson, 2003). Further, the data suggested that the zone of inhibition (mm) in heterotrophic culture condition was found to be higher as compared to the chemolithotrophic culture condition. The possible explanation may be due to the switching over of Thiobacillus ferrooxidans from chemolithotrohic to heterotrophic culture condition with comparatively less specific growth rate due to the supplement of organic carbon (glucose) leading to the formation of different organic acids as byproducts in heterotrophic culture condition (Tuttle et al., 1977) as well as cessation of chemolithotrophic growth due to increase in pH during bacterial growth.

Isolation and Characterization of Thiobacillus 81

Conclusion Coal mine AMD is a unique geomorphic system with several hostile environmental parameters, but not sterile, and support bacterial community that has physiological adaptability to thrive in such hostile condition. A detailed microbiological investigation involving complete exploration and physiological profiling of such bacteria would result in the isolation of several unique bacterial strains having bioprospecting potential. The bacterial strain i.e. Thiobacillus ferrooxidans isolated from the acidic coal mine drainage was ascertained to be Gram negative, rod shaped, aerobic, acidophilic and obligately, facultatively chemolithotrophic strain. Growth study of the bacterium under three cultural conditions revealed that its growth under chemolithotrophic condition led to more acid production. However, when glucose was available, it showed heterotrophic mode of growth with relatively faster rise of culture pH. Thus, with the availability of organic reduced carbon, the bacterium switched over to heterotrophic culture condition along with the rise of culture pH. The study suggested that if chemolithotrophic Thiobacillus ferrooxidans in mine drainage is provided with organic carbonaceous substances, it can improve the pH of AMD as a result of which other heterotrophic groups of bacteria can inhabit making it more neutral. This in other words provides a strategy for remediation of coal mine drainage to solve the crisis of aquatic pollution. The prospective environmental technology for the management of coal mine drainage to lagoon in a shallow pond, where it is to be supplemented with organic carbonaceous substances like agricultural wastes for supporting the heterotrophic growth of bacteria to make the discharge near neutral pH.

Aknowledgement Author is thankful to Prof. Niranjan Behera, School of Life Sciences, Sambalpur University, Orissa for providing laboratory facilities and constructive criticism.

References

[1] Ageeva, S. N., Kondrateva, T. F., and Karavaiko, G. I., 2001, “Phenotypic characteristics of Thiobacillus ferrooxidans strains,” Mikrobiologiya, 70, pp. 226–234. [2] Alpers, C. N., Blowes, D. W., Nordstrom D. K., and Jambor, J. L., 1994, “Secondary minerals and acid mine-water chemistry,” In: Environmental geochemistry of sulfide mine-wastes, Eds. by D. W. Blowes, and J. L. Jambor, Short Course Handbook, Vol. 22, Mineralogical Association of Canada, pp. 247-270. [3] Amaro, A. M., Chamorro, D., Seeger, M., Arredondo, R., Peirano, I, and Jerez, C. A., 1991, “Effect of external pH perturbations on in vivo protein synthesis by the acidophilic bacterium Thiobacillus ferrooxidans,” J. Bacteriol., 173, pp. 910–915. [4] Atlas, R. M., and Bartha, R., 2005, Microbial Ecology: Fundamentals & Applications, Pearson Education; 4th Eds. 82 Amiya Kumar Patel

[5] Bacelar-Nicolau, P., and Johnson, D. B., 1999, “Leaching of pyrite by acidophilic heterotrophic iron-oxidizing bacteria in pure and mixed culture,” Appl. Environ. Microbiol., 65, pp. 585–590. [6] Baker, B. J., and Banfield, J. F., 2003, “Microbial communities in acid mine drainage,” FEMS Microbiol. Ecol., 44, pp. 139–152. [7] Bond, P. L., Smriga, S. P., and Banfield, J. F., 2000, “Phylogeny of microoorgansims populating a thick, subaerial, predominantly lithotrophic biofilm at an extreme acid mine drainage site,” Appl. Environ. Microbiol., 66, pp. 3842–3849. [8] Brofft, J. E., McArthur, J. V., and Shimkets, L. J., 2002, “Recovery of novel bacterial diversity from a forested wetland impacted by reject coal,” Environ. Microbiol., 4, pp. 764–769. [9] Chavarie, C., Karamenev, D., Godard, F., Garnier, A., and Andre, G., 1993, “Comparison of kinetics of ferrous iron oxidation by three different strains of Thiobacillus ferrooxidans,” Geomicrobiology Journal, Vol. 11, pp. 57-63. [10] Chisholm, I. A., Leduc, L. G., and Ferroni, G. D., 1998, “Metal resistance and plasmid DNA in Thiobacillus ferrooxidans,” Antonie van Leeuwenhoek, 73, pp. 245–254. [11] Cravotta, C. A., Dugas, D. L., Brady, K. B. C., and Kovalchuk, T. E., 1994, “Effects of selective handling of pyritic, acid-forming materials on the geochemistry of pore gas and groundwater at a reclaimed surface coal mine in Clarion County, PA, USA,” U.S. Bur. of Mines Special Publication SP-06A, Vol. 1, pp. 365-374. [12] Edwards, K. J., Gihring, T. M., and Banfield, J. F., 1999, “Seasonal variations in microbial populations and environmental conditions in an extreme acid mine drainage environment,” Appl. Env. Microbiol., 65(8), pp. 3627-3632. [13] Edwards, K. J., Bond, P. L., Gihring, T. M., and Banfield, J. F., 2000, “Anarchaeal iron-oxidizing extreme acidophile important in acidic mine drainage,” Science, 287, pp. 1796–1798. [14] Eccleston, M., and Kelly, D. P., 1978, “Oxidation Kinetics and Chemostat Growth Kinetics of Thiobacillus ferrooxidans on Tetrathionate and Thiosulfate.,” J. Bacteriol., 134(3), pp. 718-727. [15] Ehrlich, H. L., 1990, Geomicrobiology (2nd): New York, Marcel Dekker, Inc., pp. 646. [16] Evans, D. R., and Rose, A. W., 1995, “Experiments on alkaline addition to coal mine spoil,” In: Proceedings of Sudbury '95, Mining and the Environment, pp. 49-58. [17] Evangelou, V.P., 1995, “Pyrite oxidation and its control,” CRC Press, pp. 293. [18] Ferguson, K. D., and Erickson, P. M., 1988, “Pre-Mine Prediction of Acid Mine Drainage,” In: Degraded Material and Mine Tailings, Springer-Verlag Berlin Heidelberg. [19] Friese, K., Wendt-Potthoff, K., Zachmann, D. W., Fauville, A., Mayer, B., and Veizer, J., 1998, “Biogeochemistry of iron and sulfur in sediments of an acidic mining lake in Lusatia, Germany,” Water Air Soil Poll., 108, pp. 231–247. Isolation and Characterization of Thiobacillus 83

[20] Frattini, C. J., Leduc, L. G., and Ferroni, G. D., 2000, “Strain variability and the effects of organic compounds on the growth of the chemolithotrophic bacterium Thiobacillus ferrooxidans,” Antonie van Leeuwenhoek, 77, pp. 57– 64. [21] Hallberg, K. B., and Johnson, D. B., 2001, “Biodiversity of acidophilic prokaryotes,” Adv. Appl. Microbiol., 49, pp. 37–84. [22] Hallberg, K. B., and Johnson, D. B., 2003, “Novel acidophiles isolated from moderately acidic mine drainage waters,” Hydrometallurgy, 71, pp. 139–148. [23] Harrison, J. A. P., 1985, “The acidophilic Thiobacilli and other acidophilic bacteria that share their habitat,” Annu. Rev. Microbiol., 38, pp. 265–292. [24] Hiraishi, A., Nagashima, K. V. P., Matsuura, K., Shimada, K., Takaichi, S., Wakao, N., and Katayama, Y., 1998, “Phylogeny and photosynthetic features of Thiobacillus acidophilus and related acidophilic bacteria: its transfer to the genus Acidiphilum as Acidiphilum acidophilum comb. nov.,” Int. J. Syst. Bacteriol., 48, pp. 1389–1398. [25] Hornberger, R. J., Smith, M. W., Friedrich, A. E., and Lovell, H. L., 1990, “Acid mine drainage from active and abandoned coal mines in Pennsylvania,” In: Majumdar, S.K., Miller, E.W. and Parizek, R.R., eds. Water resources in Pennsylvania--Availability, quality, and management. The Pennsylvania Academy of Science, pp. 432-451. [26] Ito, T., Nielsen, J. L., Okabe, S., Watanabe, Y., and Nielsen, P. H., 2002, “Phylogenetic identification and substrate uptake patterns of sulfate-reducing bacteria inhabiting an oxic-anoxic sewer biofilm determined by combining microautoradiography and fluorescent in situ hybridization,” Appl. Environ. Microbiol., 68, pp. 356–364. [27] Johnson, D. B., and Rang, L., 1993, “Effects of acidophilic protozoa on populations of metal-mobilizing bacteria during the leaching of pyritic coal,” J. Gen. Microbiol., 139, pp. 1417–1423. [28] Kelly, D. P., and Wood, A. P., 2000, “Reclassification of some species of Thiobacillus to the newly designated genera Acidithiobacillus gen. nov., gen. nov. and Thermithiobacillus gen. nov,” Int. J. Syst. Evol. Microbiol., 50, pp. 511–516. [29] Kleinmann, R. L. P., Crerar, D. A., and Pacelli, R. R., 1981, “Biogeochemistry of acid mine drainage and a method to control acid formation,” Mining Engineering, Vol. 33, pp. 300-303. [30] Kuenen, J. G., Robertson, L. A., and Tuovinen, O. H., 1991, “The genera Thiobacillus, Thiomicrospira, and Thiosphaera,” pp. 2638–2657. In A. Balows, H. G. Truper, M. Dworkin, W. Harder, and K.-H. Schleifer (eds.), The prokaryotes, 2nd ed., vol. 3. Springer-Verlag, New York, N.Y. [31] Leduc, L. G., and Ferroni, G. D., 1994, “The chemolithotrophic bacterium Thiobacillus ferrooxidans,” FEMS Microbiol. Rev., 14, pp. 103-120. [32] Lo´pez-Archilla, A. and Amils, R., 2001, “Microbial community composition and ecology of an acidic aquatic environment: the Tinto River, Spain,” Microb. Ecol., 41, pp. 20–35. 84 Amiya Kumar Patel

[33] McKibben, M. A., and Barnes, H. L., 1986, “Oxidation of pyrite in low temperature acidic solutions - Rate laws and surface textures,” Geochimica et. Cosmochimica Acta., Vol. 50, pp. 1509-1520. [34] Moreira, D., and Amils, R., 1997, “Phylogeny of Thiobacillus cuprinus and other mixotrophic Thiobacilli: proposal for Thiomonas gen. nov,” Int. J. Syst. Bacteriol., Vol. 47, pp. 522–528. [35] Moses, C. O., and Herman, J. S., 1991, “Pyrite oxidation at circumneutral pH,” Geochimica et. Cosmochimica Acta., Vol. 55, pp. 471-482. [36] Muyzer, G., E. C. de Waal, and Uitterlinden, A. G., 1993, “Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA,” Appl. Environ. Microbiol., 59, pp. 695–700. [37] Nordstrom, D. K., 1982, “Aqueous pyrite oxidation and the consequent formation of secondary iron minerals,” Kittrick, J. A., Fanning, D. S. and Hossner, L. R., (Eds.) Acid sulfate weathering. Soil Science Society of America, pp. 37-63. [38] Nordstrom, D. K., and Alpers, C. N., 1996, “Geochemistry of acid mine waters,” In: Plumlee, G.S. and M.J. Logsdon, eds., The Environmental Geochemistry of Mineral Deposits—Part A. Processes, methods, and health issues: Reviews in Economic Geology, Vol. 6. [39] Rawlings, D. E., 1999, “The molecular genetics of mesophilic, acidophilic, chemolithotrophic, iron- or sulfur-oxidizing microorganisms,” In Biohydrometallurgy and the Environment. Towards the Mining of the 21st Century, part B, Edited by R. Amils and A. Ballester. Amsterdam: Elsevier, pp. 3–20. [40] Rawlings, D. E., Tributsch, H., and Hansford, G. S., 1999, “Reasons why ‘Leptospirillum’-like species rather than Thiobacillus ferrooxidans are the dominant iron-oxidizing bacteria in many commercial processes for the biooxidation of pyrite and related ores,” Microbiology, 145, pp. 5–13. [41] Sand, W., 1989, “Ferric iron reduction by Thiobacillus ferrooxidans at extremely low pH values,” Biogeochemistry, 7, pp.195–201. [42] Sand, W., Rhode, K., Sobotke, B., and Zenneck, C., 1992, “Evaluation of Leptospirillum ferrooxidans for leaching,” Appl. Environ. Microbiol., 58, pp. 85–92. [43] Schleper, C., Puehler, G., Holz, I., Bambacorta, A., Janekovic, D., Santarius, U., Klenk, H. P., and Zillig, W., 1995, “Picrophilus gen. nov., fam. nov.—a novel aerobic, heterotrophic, thermoacidophilic genus and family comprising archaea capable of growth around pH 0.5,” J. Bacteriol., 177, pp. 7050–7059. [44] Schrenk, M. O., Edwards, K. J., Goodman, R. M., Hamers, R. J., and Banfield, J. F., 1998, “Distribution of Thiobacillus ferrooxidans and Leptospirillum ferrooxidans: implications for generation of acidic mine drainage,” Science, 279, pp. 1519–1522. [45] Temple, K. L., and Colmer, A. R., 1951, “The autotrophic oxidation of iron by a new bacterium: Thiobacillus ferrooxidans.,” J. Bacteriol., 62, pp. 605-611. Isolation and Characterization of Thiobacillus 85

[46] Tuovinen, O. H., and Kelly, D. P., 1974, “Studies on the growth of Thiobacillus ferrooxidans. V. Factors affecting growth in liquid culture and development of colonies on solid media containing inorganic sulphur compounds,” Arch. Microbiol., 98, pp. 351-364. [47] Tuttle, J. H., Dugan, P. R., and Apel, W. A., 1977, “Leakage of cellular material from Thiobacillus ferrooxidans in the presence of organic acids,” Appl. Env. Microbiol., 33(2), pp. 459-469. [48] Wakao, N., Koyatsu, H., Komai, Y., Sakurai, Y., and Shiota, H., 1988, “Microbial oxidation of arsenite and occurrence of arsenite oxidizing bacteria in acid mine water from a sulfur-pyrite mine,” Geomicrobiol. J., 6, pp. 11–24. [49] Watzlaf, G. R., 1992, “Pyrite oxidation in saturated and unsaturated coal waste,” In: Proceedings of the National Meeting of the American Society for Surface Mining and Reclamation, pp. 191-203. [50] Williamson, M. A., and Rimstidt, J. D., 1994, “The kinetics and electrochemical rate determining step of aqueous pyrite oxidation,” Geochimica et. Cosmochimica Acta., Vol. 58, pp. 5443-5454.

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