Free-living and surface-associated microbial communities in sand-packed columns perfused with groundwater were compared by examination of compositional and functional characteristics. The composition of the microbial communities was assessed by bulk DNA extraction, PCR amplification of 16S ribosomal DNA fragments, separation of these fragments by denaturing gradient gel electrophoresis, and sequence analysis. Community function was assessed by measurement of -glucosidase and aminopeptidase extracellular enzyme activities. Free-living populations in the aqueous phase exhibited a greater diversity of phylotypes than populations associated with the solid phase. The attached bacterial community displayed significantly greater -glucosidase and aminopeptidase enzyme activities per volume of porous medium than those of the free-living community. On a per-cell basis, the attached community had a significantly higher cell-specific aminopeptidase enzyme activity (1.07 ؋ 10 ؊7 nmol cell ؊1 h ؊1 ) than the free-living community (5.02 ؋ 10 ؊8 nmol cell ؊1 h ؊1 ). Conversely, the free-living community had a significantly higher cell-specific -glucosidase activity (1.92 ؋ 10 ؊6 nmol cell ؊1 h ؊1 ) than the surface-associated community (6.08 ؋ 10 ؊7 nmol cell ؊1 h ؊1 ). The compositional and functional differences observed between these two communities may reflect different roles for these distinct but interacting communities in the decomposition of natural organic matter or biodegradation of xenobiotics in aquifers.Functional differences between free-living and particle-or macroaggregate-associated bacteria have been documented in the water columns of aquatic (14, 31, 37) and marine (20,32,35,39,40) environments. Because cell attachment is known to strongly but unpredictably affect cell physiology (29, 41), there has been uncertainty whether these functional differences are due to changes in functional expression or differences in community composition (12,22,32,35). Independent reports of compositional differences in free-living and attached bacteria in marine water columns (1,5,12) and two studies in which aspects of both structure and function of these two communities were measured (10, 35) indicate that compositional differences may be responsible (in part) for the observed functional differences.The current working model in aquatic and marine environments is that attached and unattached bacteria comprise two distinct but interacting communities, shaped by differential access to nutrients and susceptibility to predation (14,20,23,31,32,35,39,40). For example, bacteria attached to particulate matter suspended in water columns display greater extracellular enzyme activity than their free-living counterparts (20,22,31,32,35,39). However, uptake by the attached populations of the products of this extracellular activity appears to be uncoupled from the enzyme activity, creating a supply of usable organic carbon to free-living bacteria in the surrounding water (22,31,39).Differences in rates of polymer hydrolysis, substrate u...