The way that gaseous metabolite production changes along with biofilm architecture development is poorly understood. To address this question, we developed a novel flow reactor biofilm culture method that allows for simultaneous assessment of gaseous metabolite production and architecture visualization. In this report, we establish the utility of this method using denitrification by Pseudomonas aeruginosa biofilms as a model system. Using this method, we were able to collect and analyze gaseous metabolites produced by denitrification and also visualize biofilm architecture in a nondestructive manner. Thus, we propose that this novel method is a powerful tool to investigate potential relationships between biofilm architecture and the gas-producing metabolic activity of biofilms, providing new insights into biofilm ecology.It is known that environmental biofilms often produce gaseous metabolites. For example, natural lake reed biofilms are reported to produce N 2 or N 2 O (28). Furthermore, microbial biofilms are utilized in wastewater treatment and other industrial processes to convert soluble material into gaseous material, such as the conversion of nitrate into N 2 or N 2 O by denitrification (2,21,30). Previous studies of biofilm architecture have reported correlations between metabolic substrates and biofilm architecture (11,24,27), and another study showed the importance of biofilm structures in the distribution of dissolved gases (4). However, such correlations between biofilm architecture and gaseous metabolite production remain poorly understood.If one were able to show simultaneously occurring changes in architecture and gaseous metabolite production, the existence of a correlation between biofilm architecture and gaseous metabolite production would be indicated. To date, the analysis of gaseous metabolite production by biofilms has mainly been performed using test tubes or flasks sealed with butyl rubber stoppers (28). These airtight containers allow for collection of gaseous metabolites and are suitable for batch culture experiments. However, there are currently no nondestructive methods to simultaneously allow for both biofilm visualization and gaseous metabolite analysis. Such a method would provide information on how gaseous metabolite production activity changes along with biofilm development.The flow reactor method (15, 18), combined with confocal laser scanning microscopy (CLSM), is preferred in studies of biofilm architecture and development. Both electron microscopy and CLSM are often used in visualizing biofilms (12); however, electron microscopy requires dehydration (and therefore termination of biofilm growth), which in some cases distorts or damages bacterial cells (7). In contrast, CLSM can be used to visualize the three-dimensional architecture of living biofilms and can be applied to the flow reactor method while maintaining continuous culture (15,18). If collection of gaseous metabolites is achieved in a flow reactor system, both biofilm visualization and gaseous metabolite analysis can...