This dissertation describes several studies of anaerobic benzene degradation by microorganisms and fulfills four objectives. The first and second objectives were to characterize microbial populations in two benzene-degrading consortia (one methanogenic and one nitrate-reducing) on a physiological and molecular level. The shortest doubling times, 8 to 9 days, were observed in nitrate-reducing cultures. The highest substrate concentration utilized, 1000 μm, and maximum absolute rates of benzene degraded, 75 μm/day, were observed in methanogenic cultures. Five Bacterial 16S rRNA sequences, one of which resembled a clone previously found in a sulphate-reducing benzene-degrading culture, were identified in the methanogenic culture. Four Bacterial and no Archaeal 16S rRNA sequences were identified in the nitrate-reducing culture. One clone comprised 70% of the culture and was phylogenetically 93% similar to both Azoarcus and Dechloromonas species.

The third objective was to isolate and characterize pure cultures capable of anaerobic benzene biodegradation. Two co-cultures called Dechloromonas co-culture (DCC) and Azoarcus co-culture (ACC) were successfully isolated from a nitrate-reducing enrichment culture. DCC is ~99% Dechloromonas sp. and ~1% an unidentified eubacterium clone. ACC is ~98% Azoarcus sp. and ~2% Pseudomonas sp. Both co-cultures were capable of anaerobic benzene, toluene and benzoate degradation under nitrate-reducing conditions. The isolated Pseudomonas sp. was capable of aerobic benzene degradation and hydrogen utilization under nitrate-reducing conditions. The isolated unidentified eubacterium clone did not exhibit growth on any of the substrates tested. The Dechloromonas and Azoarcus organisms are responsible for anaerobic benzene degradation.

The fourth objective was to identify the key metabolic steps in the anaerobic benzene degradation pathways. ¹³C₆-benzene experiments were conducted with the methanogenic and nitrate-reducing enrichments. Under methanogenic conditions a maximum of 8 μm ¹³C₆-phenol and 3 μm ¹³C₆-benzoate were detected. These results indicate that under methanogenic conditions a pathway where benzene undergoes hydroxylation to phenol and further transformation to benzoate exists. Under nitratereducing conditions a maximum of 90 nM ¹³C₆-toluene, 120 nM ¹³C₆-benzoate and no phenol were detected as intermediates of benzene degradation. This is the first evidence for a pathway where benzene undergoes methylation to toluene and further transformation to benzoate.