Ambient temperature freshwater carbonates ("tufas") have outstanding potential as palaeoclimate archives. However, it has proven difficult to constrain the behaviour of the geochemical parameters on which important proxy systems rely. Current thinking on the development of trace element ratios in tufa rests upon an assumption of precipitation driven by degassing of CO2, with the final precipitate being in equilibrium with ambient river water. This study provides evidence that although supersaturation is largely regulated by degassing, precipitation in sterile flowing systems does not occur throughout the air-water interface, but only at lines of intersection between this interface and a solid surface. Precipitation at the bottom, as is normal in river sites, is not found to occur. However, when benthic microbial communities ("biofilms") are present, extensive precipitation does occur within them in preference and/or addition to that at the air-water interface. Precipitation is therefore not purely a consequence of degassing, and some microbiological influence is necessary to generate precipitates analogous to nature. We further investigate whether these biofilms alter the behaviour of trace elements within tufa systems, in addition to modulating the sites and mechanisms of precipitation, via a series of experiments. Biofilms are shown to accumulate large inventories of calcium and other dissolved ions (Ba, Sr, and Mg), and we demonstrate that this process is chemoselective in favour of ions with low charge densities. These observations have consequences for interpretation of trace element records from tufas based on an assumption of equilibrium with river water, and also for other carbonate deposits associated with biofilm activity (travertines, marine stromatolites, lacustrine and soil carbonates, speleothems). Conversely, our study provides new insight into the reasons for discrepancy of tufa Mg/Ca ratios from expectation, and thus provides encouragement for the potential of Mg/Ca palaeothermometry in freshwater carbonate systems.