Abstract: A mixed aerobic-anaerobic microbial treatment process was developed previously for acid mine drainage (AMD) using straw as a substrate. The process was effective only if AMD was supplemented with sucrose. The present study was conducted to determine which, if any, of three cellulosic materials could sustain the microbial treatment of AMD without the addition of a sucrose amendment and to determine the effect of the retention time on the performance of the reactors. The performance of small reactors that treated simulated AMD in the continuous mode was evaluated using alfalfa (Medicago sativa L.) hay, timothy (Phleum pratense L.) hay, and straw with a 5 d retention time. Parameters measured were pH, Fe, Al, sulfate, and ammonium. Timothy hay and straw sustained AMD mitigation for 3 wk, and thereafter all activity ceased. After the reactors ceased treating AMD, the mitigative activities were reinitiated by the addition of sucrose, but not by urea. Alfalfa sustained AMD mitigation for a longer time period than either straw or timothy. The effect of three retention times, 3.5, 7, and 35 d, was then investigated for reactors containing fresh alfalfa. Increasing the retention time resulted in better metal removal and a greater pH increase. With a 7-d retention time, 75 L of simulated AMD were neutralized from a pH of 3.5 to a pH value greater than 6.5. Reactors operating with a 3.5-d retention time treated only 58.3 L of simulated AMD before failing. Ammonium was detected in effluents of active reactors. The results of this study indicate that a low maintenance microbial treatment system can be developed with alfalfa as a substrate without the addition of a sucrose amendment.