High-performance MS instrumentation coupled with improved protein extraction 
techniques enables metaproteomics to identify active members of soil and 
groundwater microbial communities. Metaproteomics workflows were applied to 
study the initial responses (i.e. 4 days post treatment) of the indigenous 
aquifer microbiota to biostimulation with emulsified vegetable oil (EVO) at a 
uranium-contaminated site. Members of the Betaproteobacteria (i.e. 
Dechloromonas, Ralstonia, Rhodoferax, Polaromonas, Delftia, Chromobacterium) and 
the Firmicutes dominated the biostimulated aquifer community. Proteome 
characterization revealed distinct differences between the microbial biomass 
collected from groundwater influenced by biostimulation and groundwater 
collected upgradient of the EVO injection points. In particular, proteins 
involved in ammonium assimilation, EVO degradation, and polyhydroxybutyrate 
granule formation were prominent following biostimulation. Interestingly, the 
atypical NosZ of Dechloromonas spp. was highly abundant, suggesting active 
nitrous oxide (N2 O) respiration. c-Type cytochromes were barely detected, as 
was citrate synthase, a biomarker for hexavalent uranium reduction activity, 
suggesting that uranium reduction has not commenced 4 days post EVO amendment. 
Environmental metaproteomics identified microbial community responses to 
biostimulation and elucidated active pathways demonstrating the value of this 
technique as a monitoring tool and for complementing nucleic acid-based 
approaches.