Chemolithoautotrophic sulfate reduction (CSR) was tested to treat natural acid mine drainage influenced groundwaters. The long term behavior was studied for more than 3 years under groundwater conditions (10 degrees C, autochthonous sulfate reducing bacteria (SRB)) without biomass replenishment in a 190 L bench scale reactor. The process produces water with alkalinity >10 mM. pH can be controlled by p(CO2) for all expectable water qualities. SRB were immobilized using an expanded clay bed. After 1.3 years of operation, a constant biomass content and sulfate reduction rate of 0.25-0.30 mmol(SO4) (L h)(-1) were established. The sulfate reduction rate was limited by biomass content. Most of the electrons were used for sulfate reduction (98%). The hydrogen turn over in competing processes like methanogenesis and homo-acetogenesis was successfully suppressed by adjusting the sulfate concentration to be >2 mM in the runoff. (C) 2011 Elsevier Ltd. All rights reserved.