Anaerobic sludge were cultured and acclimated by adding different co-metabolism substrates under six operating conditions, using chloroform as model contaminant in an anaerobic environment. The microorganisms obtained with chloroform biodegradability were concentrated and cultured before adding into the simulated decontaminating apparatus. The results showed that the chloroform could be degraded by the microorganisms under the anaerobic condition, and the addition of co-metabolism substrates could improve the biodegradation efficiency. Moreover, the biodegradation efficiency varied with different co-metabolism matrix. The removal efficiency of pollutants could reach 75% using the microorganisms acclimated with glucose and methanol as co-metabolism substrates.