Riboflavin- and cobalamin-mediated biodegradation of chloroform in a methanogenic consortium

Chloroform (CF) is an important priority pollutant contaminating groundwater. Reductive dechlorination by anaerobic microorganisms is a promising strategy towards the remediation of CF. The objective of this study was to evaluate the use of redox active vitamins as electron shuttles to enhance the anaerobic biodegradation of CF in an unadapted methanogenic consortium not previously exposed to chlorinated compounds. Only negligible degradation of CF was observed in control cultures lacking redox active vitamins. The addition of riboflavin (RF), cyanocobalamin (CNB₁₂), and hydroxycobalamin (HOB₁₂) enabled biodegradation of CF. The reactions were predominantly catalyzed biologically as evidenced by the lack of any CF conversion in heat-killed controls amended with the cobalamins or minor conversion with RF. In live cultures, significant increases in the rate of CF conversion was observed at substoichiometric molar ratios as low as 0.1 to 0.01 vitamin:CF for RF and CNB₁₂, respectively. At the highest molar vitamin:CF ratios tested of 0.2, the first-order rate constant of CF degradation was 5.3- and 91-fold higher in RF and CNB₁₂ amended cultures, respectively, compared to the unamended control culture. The distribution of biotransformation products was highly impacted by the type of redox active vitamin utilized. Cultures supplemented with RF provided high yields of dichloromethane (DCM). On the other hand, cobalamins promoted the near complete mineralization of organochlorine in CF to inorganic chloride and lowered the yield of DCM. In cultures where no or little CF bioconversion occurred, prolonged exposure to CF resulted in cell lysis, as evidenced by the release of intracellular chloride. The results taken as a whole suggest that the anaerobic bioremediation of CF-contaminated sites can greatly be improved with strategies aimed at increasing the concentration of redox active vitamins.