Investigation of cell exudates active in carbon tetrachloride and chloroform degradation

Contamination of groundwater by chlorinated solvents such as carbon tetrachloride (CCl₄) and chloroform (CHCl₃) is a widespread problem. The cell exudates from the methanogen Methanosarcina thermophila are active in the degradation of CCl₄ and CHl₃. This research was performed to characterize these exudates. Examination of the influence of pH indicated that activity was greater under alkaline conditions. Rapid CCl₄ degradation occurred from 35-65°C, with first-order degradation rate coefficients increasing as temperature increased. It was found that proteins were not responsible for CCl₄ degradation. The active agents in the cell exudates were <10 kDa in size, with degradation activity present in both 1-10 kDa and <1 kDa size ranges. Upon purification of the <10 kDa size range of the cell exudates on a C₁₈ chromatography column, 17 fractions (out of 100) degraded >50% of the added CCl₄ in 8 h. These 17 fractions were pooled into three samples based on their elution time from the C₁₈ column. One of these pooled samples contained elevated levels of cobalt, zinc, and iron, at 2, 3, and 13 times the levels measured in similarly fractionated and pooled samples of medium, respectively. The UV-visible spectrum of this pooled sample had an absorption maximum at 560-580 nm, which is similar to the absorption maxima of heme (approximately 550 and 575 nm). The two other pooled samples contained elevated levels of zinc at 11 and 22 times the concentration measured in similarly fractionated and pooled samples of medium, respectively, and also contained very Iow levels of nickel, cobalt, and iron. This research suggests that the cell exudates from M. thermophila contain porphorinogen-type molecules capable of dechlorination, possibly excreted corrinoids, hemes, and zinc-containing molecules.