Nitrogen isotope records of chloropigments can be used to reconstruct photic zone processes. However, the 15 N offset between chlorophyll and biomass (ε por =δ 15 N biomass −δ 15 N chlorophyll ) of cyanobacteria and eukaryotes grown in laboratory batch cultures differs significantly and the cause of the difference remains unknown. Here, in four experiments with Synechocystis sp. PCC 6803, values of ε por were statistically invariant when cultures were maintained at pH 6.5–9.5 under constant growth conditions in a chemostat. The results suggest that the negative values of ε por observed for freshwater cyanobacteria are not artifacts of batch culturing.