Coal-generated acid mine drainage (AMD) is characterized by low-pH waters with excessive loads of dissolved species such as SO4, Fe, Al and Mn along with other elements of environmental concern (i.e. Cd, As, Cr, Ni, Pb, Se and Cu). To mitigate this problem, anaerobic sulfate reducing bioreactors (ASRB) have been implemented as a technology for passive treatment systems that utilize low-cost organic substrates to stimulate biologically enhanced contaminant sequestration. Previous work has identified the establishment of diverse microbial communities in which a hierarchal chain of substrate degradation processes is essential in developing sustainable environments to produce long-lived sulfate-reducing microbial (SRM) populations. In this study, to determine the optimal mixture of substrate types, alternating ratios of herbaceous (ie. leaves, grass, spent brewing grains) and ligneous (i.e. maple wood chips and saw dust) reactor matrices were tested. Five bioreactors along with one control reactor containing only limestone were constructed at the Tab-Simco abandoned mine land (AML) site in southern Illinois, USA. The field experiments were monitored over ~ one year (377 days) to evaluate the physical, geochemical and microbiological parameters which dictate ASRB efficiency in remediation of AMD contaminants. Results from this experiment documented contaminant removal in all reactors. However, the bioreactors established SRM populations that contributed to enhanced removal of SO4, Fe, and trace metals (i.e. Cu, Cd, Zn, Ni). Geochemical assessment of the aqueous environments established within the bioreactors suggested multiple pathways of contaminant sequestration. This included the formation of Fe-oxyhydroxide precipitates, adsorption, co-precipitation (e.g. Zn/Ni-Ferrites) and bio-induced sulfide mineralization. Activity of the SRMs was dependent on temperature, with bioreactors exhibiting decreases in both effluent sulfide concentrations and 34S-depletion of sulfate during low-T months (i.e. T < 10°C). Overall, maximum remediation of dissolved constituents SO4, Fe, Al and Mn was obtained in the predominantly herbaceous bioreactors. Extrapolation of our results to the full-scale Tab-Simco bioreactor indicated that, over the course of one year, the herbaceous bioreactors would remove ~75,600 kg SO4, 21,800 kg Fe, 8000 kg Al, and 77 kg Mn. This represents a 21.7 wt%, 41.5 wt%, 9.4 wt% and 81.8 wt% increase in SO4, Fe, Al and Mn removal over dominantly ligneous bioreactors, respectively. Although the overall Fe removal within the limestone control reactor reached 44.5 mol%; removal of 19.5 mol% SO4 and 36.9 mol% Al from influent AMD were significantly less when compared to the bioreactors. These results imply that ASRB technologies are promising in remediation of coal-generated AMD and increasing herbaceous content of bioreactors can significantly enhance contaminant sequestration. However, geochemical results also displayed seasonal variation in redox gradients within our field ASRB's which may induce dissolution of the redox sensitive phases produced within bioreactors. Furthermore, optimal microbial-mediated sulfate reduction may be inhibited by the high surface areas of the abundant Fe/Al-oxyhydroxides which dominate the system. Therefore, to enhance ASRB remediation capacity, future designs must optimize not only the organic carbon substrate but also include a pretreatment phase in which the bulk of dissolved Fe/Al-species are removed from the influent AMD prior to entering the bioreactor.