Tufas represent a palaeoclimatic archive of potentially global significance. However, uncertainty remains over the exact process of calcite precipitation from these systems, inhibiting our ability to decipher the precise meaning of geochemical records. For example, field studies of alkaline stream systems are unable to disentangle the influence of temperature and photosynthesis on ambient hydrochemistry on diurnal and annual timescales. This report describes a series of flume experiments in which temperature and light conditions are manipulated separately. These experiments reveal that precipitation of calcite occurs preferentially under conditions of rising pH, and consequently at the night-day transition. The amplitude of diurnal changes is regulated by the buffering capacity (i.e. alkalinity) of the ambient water and by the daytime balance of photosynthesis and respiration. Respiration is shown to be strongly affected by temperature, whereas photosynthesis is found to be limited by nutrient and/or DIC availability making temperature impacts minor. Consequently, macroenvironment pH during both day and night-time tend to be higher under lower temperatures, in contrast to expectation. These observations may have potential implications for the isotopic geochemistry of tufa carbonate, promoting slightly lower δ18O, due to the carbonate ion effect, and more significantly negative δ13C, due to incorporation of respired CO2 accumulated during the night. The observation that long periods of daylight are not necessarily needed for photosynthetically induced precipitation to occur confirm previous arguments that seasonal lamination requires either strong variability in ambient physicochemical activity or an ecological change in the microbial assemblage, and cannot be ascribed to reduced temperature and light intensity.