This thesis describes the characterization of benzene-degrading denitrifying cultures. Four objectives were pursued. The first objective was to identify conditions that promote or inhibit benzene decomposition and thus, to improve the biodegradation capacity of the cultures. FeS, resazurin, and nitrite had a detrimental impact on benzene degradation, whereas addition of supernatant from an active culture improved the benzene degradation activity by reducing the lag times. The second objective was to determine the microbial community composition in enrichment cultures and to identify the bacterial species that mediate benzene mineralization. Five dominant bacterial Operational Taxonomic Units (OTUs) were identified. The most abundant phylotype was related to the gram-positive Peptococcaceae family. Other bacteria present were closely affiliated with Dechloromonas, Azoarcus, Chlorobi and Anammox species. To correlate the growth of these specific microbes with benzene degradation, the abundance of specific 16S rRNA genes was monitored during mineralization process using quantitative polymerase chain reaction (qPCR). Based on the result of qPCR experiments and information about the metabolisms of the above bacteria, a syntrophic mode of benzene degradation was hypothesized to occur under denitrifying conditions. In this process, Peptococcaceae initiate attack on benzene, and ferment benzene to hydrogen and low molecular weight products such as acetate. These products are then consumed by nitrate-respiring Azoarcus and Dechloromonas or phototrophic Chlorobi. Anammox bacteria recycle and detoxify nitrite, and stabilize the culture. The third objective was to isolate and characterize pure cultures with the ability to mineralize benzene anaerobically. Dechloromonas- and Dechlorosoma-like microorganisms were isolated from several benzene-degrading microcosms. Theses bacteria, however, were not able to metabolize benzene anaerobically. The fourth objective was to investigate the key metabolic steps in the anaerobic benzene degradation pathway and to identify enzymes that are involved in this process. Differential transcription during growth of the culture on benzene versus growth on a metabolite of benzene degradation, i.e. benzoate was examined. Carboxylase-related genes were specifically transcribed in the presence of benzene. Furthermore, mRNA sequences corresponding to the genes that encode different enzymes of the benzoyl-CoA degradation pathway were present in the culture. These findings suggest that mineralization of benzene starts by its activation to benzoate through a carboxylation reaction catalyzed by benzene carboxylase. Benzoate is further metabolized through benzoyl-CoA pathway.

This thesis describes the characterization of benzene-degrading denitrifying cultures. Four objectives were pursued. The first objective was to identify conditions that promote or inhibit benzene decomposition and thus, to improve the biodegradation capacity of the cultures. FeS, resazurin, and nitrite had a detrimental impact on benzene degradation, whereas addition of supernatant from an active culture improved the benzene degradation activity by reducing the lag times. The second objective was to determine the microbial community composition in enrichment cultures and to identify the bacterial species that mediate benzene mineralization. Five dominant bacterial Operational Taxonomic Units (OTUs) were identified. The most abundant phylotype was related to the gram-positive Peptococcaceae family. Other bacteria present were closely affiliated with Dechloromonas, Azoarcus, Chlorobi and Anammox species. To correlate the growth of these specific microbes with benzene degradation, the abundance of specific 16S rRNA genes was monitored during mineralization process using quantitative polymerase chain reaction (qPCR). Based on the result of qPCR experiments and information about the metabolisms of the above bacteria, a syntrophic mode of benzene degradation was hypothesized to occur under denitrifying conditions. In this process, Peptococcaceae initiate attack on benzene, and ferment benzene to hydrogen and low molecular weight products such as acetate. These products are then consumed by nitrate-respiring Azoarcus and Dechloromonas or phototrophic Chlorobi. Anammox bacteria recycle and detoxify nitrite, and stabilize the culture. The third objective was to isolate and characterize pure cultures with the ability to mineralize benzene anaerobically. Dechloromonas- and Dechlorosoma-like microorganisms were isolated from several benzene-degrading microcosms. Theses bacteria, however, were not able to metabolize benzene anaerobically. The fourth objective was to investigate the key metabolic steps in the anaerobic benzene degradation pathway and to identify enzymes that are involved in this process. Differential transcription during growth of the culture on benzene versus growth on a metabolite of benzene degradation, i.e. benzoate was examined. Carboxylase-related genes were specifically transcribed in the presence of benzene. Furthermore, mRNA sequences corresponding to the genes that encode different enzymes of the benzoyl-CoA degradation pathway were present in the culture. These findings suggest that mineralization of benzene starts by its activation to benzoate through a carboxylation reaction catalyzed by benzene carboxylase. Benzoate is further metabolized through benzoyl-CoA pathway.