Biofilms are important in aquatic nutrient cycling and microbial proliferation. In these structures, nutrients like carbon are channeled into the production of extracellular polymeric substances or cell division; both are vital for microbial survival and propagation. The aim of this study was to assess carbon channeling into cellular or noncellular fractions in biofilms. Growing in tubular reactors, biofilms of our model strain Pseudomonas sp. strain CT07 produced cells to the planktonic phase from the early stages of biofilm development, reaching pseudo steady state with a consistent yield of approximately 10(7) cells.cm(-2).h(-1) within 72 h. Total direct counts and image analysis showed that most of the converted carbon occurred in the noncellular fraction, with the released and sessile cells accounting for <10% and <2% of inflowing carbon, respectively. A CO(2) evolution measurement system (CEMS) that monitored CO(2) in the gas phase was developed to perform a complete carbon balance across the biofilm. The measurement system was able to determine whole-biofilm CO(2) production rates in real time and showed that gaseous CO(2) production accounted for 25% of inflowing carbon. In addition, the CEMS made it possible to measure biofilm response to changing environmental conditions; changes in temperature or inflowing carbon concentration were followed by a rapid response in biofilm metabolism and the establishment of new steady-state conditions.