This investigation evaluated production of extracellular polysaccharides (EPS) from the decomposition of cotton gin trash (CGT) in aerobic, anaerobic and continuous percolating environments. A further variation of pre-rinsing CGT was investigated in order to determine if the pollution potential from CGT could be minimized. Since previous investigations have used a colorimetric method for quantifying EPS that yields varying results when the constituent sugars are at different molar concentrations, a gravimetric method was used to quantify EPS production. The accumulation of uronides in the bound EPS suggests that the bound portion is critical in seal formation. The anaerobic environment showed an initial increase of EPS to 3 mg/g CGT and then maintained a constant level. The aerobic environment produced 9 mg of EPS/g CGT after 26 days with a continuing upward trend. The continuous percolating environment had concentrations similar to the aerobic environment and showed decreased hydraulic conductivity with increased EPS.