Assessing the suitability of Imaging-PAM fluorometry for monitoring growth of benthic diatoms

Abstract

High-throughput monitoring of growth using Pulse Amplitude Modulated (PAM) chlorophyll fluorescence imaging (Imaging-PAM) is an important tool for experimental ecological and physiological research on benthic diatom cultures, as due to substrate adhesion non-destructive subsampling for biomass determination is difficult and/or not representative. We evaluated the reliability of Imaging-PAM for quantifying benthic diatom biomass by comparing biomass determinations based on PAM fluorescence parameters to those based on cells counts and chlorophyll a measurements for cultures of the diatoms Cylindrotheca closterium and Seminavis robusta in different growth phases (exponential vs stationary) and cell size ranges. We found that PAM fluorescence imaging is particularly suited to monitor growth during the exponential phase. The dark adapted minimal fluorescence (F0) parameter proved to be the most reliable fluorescence-based biomass proxy, especially for establishing complete growth curves, as it was the most robust parameter to growth phase changes. A dark adaptation period prior to the PAM measurements improved the quantification of the stationary but not of the exponential cultures. Under the right conditions, dark adaptation can thus be omitted for exponentially growing cultures to enhance throughput. The use of multiwell plates for growth monitoring with the Imaging-PAM is feasible even though there can be a slight biomass underestimation near of the edges of the plate. Based on these findings, we formulate specific guidelines for using Imaging-PAM fluorometry in experiments with benthic diatom cultures. We especially recommend taking caution when working under conditions which potentially influence the condition of the photosynthetic apparatus of the algae.