The influence of hard substratum reflection and calibration profiles on in situ fluorescence measurements of benthic microalgal biomass.

Measuring chlorophyll-a fluorescence is a commonly used method to determine microphytobenthic biomass expressed as chlorophyll-a per square centimetre. However, this in situ method is affected by reflection from the substratum which triggers an additional fluorescence signal within the microphytobenthic biofilm. Depending on the colour and texture of the natural substratum, this effect can lead to a considerable overestimation of microphytobenthic biomass. The results cannot be corrected for this effect by performing an auto-zero measurement, since the overestimation is not caused by an offset of the fluorometer. This article describes a substratum-specific correction procedure using a 700 nm signal to eliminate this effect by quantifying the fluorescence signal as a result of the reflection. An empirical relationship between the 700 nm signal and the additional fluorescence is used to calculate a correction factor for the reflective properties of the substratum. The factor is determined and applied during each biomass measurement, thereby making an additional calibration step for each individual type of substratum superfluous. This new method improves the reliability of the results significantly without increasing the time necessary to perform the measurements and without complicating the measurement procedure.