• benzene degradation;
  • compound-specific stable isotope analysis;
  • protein-SIP/RNA-SIP;
  • stable isotope probing


We identified phylotypes performing distinct functions related to benzene degradation in complex microbial biofilms from an aerated treatment pond containing coconut textile. RNA- and protein-stable isotope probing (SIP) and compound-specific stable isotope analysis were applied to delineate bacteria and predominant pathways involved in the degradation of benzene. In laboratory microcosms, benzene was degraded at rates of ≥ 11 μM per day and per gram coconut textile under oxic conditions. Carbon isotope fractionation with isotopic enrichment factors (ε) of −0.6 to −1‰ and no significant hydrogen isotope fractionation indicated a dihydroxylation reaction for the initial ring attack. The incubation with [13C6]-benzene led to 13CO2 formation accompanied by 13C-labeling of RNA and proteins of the active biomass. Phylogenetic analysis of the 13C-labeled RNA revealed that phylotypes related to Zoogloea, Ferribacterium, Aquabacterium, and Hydrogenophaga within the Betaproteobacteria predominantly assimilated carbon from benzene. Although the phylogenetic classification of identified 13C-labeled proteins was biased by the incomplete metagenome information of public databases, it matched with RNA-SIP results at genus level. The detection of 13C-labeled proteins related to toluene dioxygenase and catechol 2,3-dioxygenase suggests benzene degradation by a dihydroxylation pathway with subsequent meta-cleavage of formed catechol.