Mining activities, particularly coal mining, lead to the excavation of large volumes of pyrite rich soils. When exposed to air (oxygen) and water these pyrite complexes are oxidised to form highly acidic and corrosive wastewaters collectively termed acid mine drainage (AMD). Containing elevated levels of sulphates (SO42-) and toxic dissolved heavy metals, AMD seeping from mining sites, active or abandoned, poses a major environmental risk to aquatic bio-systems – not only in South Africa but globally. Chemical neutralization of AMD is expensive and often challenging. Biological sulphate reduction provides a promising and cheaper alternative to the treatment of sulphate rich wastewaters. Little, however, is known about the microbial communities involved in biological treatment systems and the effect of external factors thereon. Studying microorganisms in their natural environment is extremely difficult. The limitations of culture-based methods only provide a limited insight into the bacterial diversity of natural habitats and the microbial communities present. With the progressive advances in molecular biology, non culture-based tools such as DGGE, FISH and more recently t-RFLP allow easier and much more accurate studies of microbial communities within their natural as well as man-made environments. This study specifically investigated the use of t-RFLP to study microbial communities and dynamics within sulphate removing bioreactors. The set up and optimization of a t-RFLP system to specifically study microbial communities from sulphate removing bioreactors were investigated and the applicability of t-RFLP demonstrated. Copyright / Dissertation (MSc)--University of Pretoria, 2009. / Microbiology and Plant Pathology / unrestricted