The slow-release, fermentable, organic substrates commonly used to support the biological treatment of mine impacted water (MIW) are often deficient in nitrogen, thereby limiting the activity of sulfate reducing bacteria and inhibiting the performance of passive treatment systems. Recently, our laboratory has shown that chitin (poly-N-acetyl-glucosamine), a nitrogen-rich, sustainable waste product of the shellfish industry, is capable of enhancing the activity of sulfate reducing bacteria and improving the efficiency of MIW treatment. This research explores the possibility of using crab-shell chitin as a fractional amendment to spent mushroom compost substrate (SMS) to facilitate the development of a cost-effective, practical approach for thorough MIW bioremediation. Continuous-flow laboratory columns were used to evaluate substrate mixtures for their ability to support bacterial diversity and contaminant reduction during treatment of acidic MIW. Five PVC columns with lateral sampling ports were established to evaluate the following substrate conditions: no substrate (inert sand used to fill column); 10% limestone and 90% SMS; 5% crab-shell chitin and 95% SMS; 50% crab-shell chitin and 50% SMS; 100% crab-shell chitin. A mixture of substrate, sand (to provide sufficient hydraulic conductivity), and sediment freshly collected from a local stream (as a bacterial source) were packed into the columns. Column performance was evaluated at 6-, 12-, and 24-hour hydraulic residence times to assess substrate suitability for a field-scale passive treatment system. Aqueous samples were collected regularly from the column influent, effluent, and lateral sampling ports. These samples were tested for pH, acidity, alkalinity, volatile fatty acids, chemical oxygen demand, ammonium, anions, dissolved metals, and oxidation/reduction potential (ORP). All substrates effectively neutralized acidic MIW and reduced sulfate at the start of the experiment. Columns were operated until substrate exhaustion in order to provide an estimate of substrate longevity. The treatment capacity of each substrate was defined here as the volume of MIW treated to pH>6 and alkalinity >0 mg/L as CaCO3, prior to the breakthrough of metals to influent levels. The treatment capacity (and associated substrate costs) for each substrate mixture was determined to be: 36.7 L/kg ($1.38/1000 L) for 10% limestone + 90% SMS; 40.1 L/kg ($2.95/1000 L) for 5% chitin + 95% SMS; 162 L/kg ($4.25/1000 L) for 50% chitin + 50% SMS; and 428 L/kg ($3.09/1000 L) for 100% chitin. Based on the calculated treatment capacities as well as effluent water quality data over the lifetime of the columns, it appears that a small fraction of chitin (5%) does not provide a significant benefit over traditional limestone and compost substrates. Larger fractions of chitin (50-100%) are significantly more efficient than traditional limestone substrates, especially for the removal of metals.