Current knowledge of the physiology and phylogeny of polycyclic aromatic 
hydrocarbon (PAH) degrading bacteria often relies on laboratory enrichments and 
isolations. In the present study, in situ microcosms consisting of activated 
carbon pellets (BACTRAP®s) were loaded with either (13) C-naphthalene or (13) 
C-fluorene and were subsequently exposed in the contaminant source and plume 
fringe region of a PAH-contaminated aquifer. Metaproteomic analysis and 
protein-stable isotope probing revealed Burkholderiales, Actinomycetales, and 
Rhizobiales as the most active microorganisms in the groundwater communities. 
Proteins identified of the naphthalene degradation pathway showed a relative 
(13) C isotope abundance of approximately 50 atom% demonstrating that the 
identified naphthalene-degrading bacteria gained at least 80% of their carbon by 
PAH degradation. Although the microbial community grown on the fluorene-BACTRAPs 
showed a structure similar to the naphthalene-BACTRAPs, the identification of 
fluorene degraders and degradation pathways failed in situ. In complementary 
laboratory microcosms, a clear enrichment in proteins related to Rhodococcus and 
possible fluorene degradation enzymes was observed. This result demonstrates the 
impact of laboratory conditions on microbial community structure and activity of 
certain species and underlines the need on in situ exploration of microbial 
community functions. In situ microcosms in combination with protein-stable 
isotope probing may be a significant tool for in situ identification of 
metabolic key players as well as degradation pathways.