Citation:

Katona, L., Y. Vadeboncoeur, C. Nietch, AND K. Hossler. A Novel Thin-Film Technique to Improve Accuracy of Fluorescence-Based Estimates for Periphytic Biofilms. WATER. MDPI, Basel, Switzerland, 13(11):1464, (2021). https://doi.org/10.3390/w13111464

Impact/Purpose:

Pulse-amplitude modulated (PAM) fluorometry has emerged as a vital tool to assess physiological efficiencies of photosynthesizing organisms and communities. The technique is both rapid and non-destructive and measures chlorophyll fluorescence in response to increasing levels of irradiance. Various studies, however, have demonstrated theoretically and empirically, that estimates of the photophysiological parameters are subject to overestimation when the photosynthesizing sample has depth or optical density (e.g. epilithic biofilm, microphytobenthos, thick plant tissue). In addition to misestimation, the effect of depth integration has the potential to confound bioassays or similar impact studies where biofilm thickness might vary with condition. We present an alternative technique to improve estimation accuracy of photophysiological parameters particularly for periphytic biofilms. With this technique, a vertically-representative subsample of a biofilm is spread evenly on a microscope slide, thus creating a thin-film layer which is then assessed using a conventional PAM fluorometer. The thin-film ensures that the entire subsample will receive the applied irradiance, rather than some portions receiving attenuated irradiance. The proposed technique is relatively easy to implement with a standard PAM fluorometer and is suitable for measurements in the lab or in the field.

Description:

Recent studies suggest that photophysiological parameters for intact substrates with depth (e.g., periphytic biofilms, microphytobenthos) are overestimated by pulse-amplitude modulated (PAM) fluorometry. This overestimation results from depth-integration effects, following the activation of deeper photosynthesizing layers by an attenuated light signal. To mitigate this error, we propose a novel slide-based thin-film technique in which fluorescence is measured on a vertically representative subsample of the biofilm, spread evenly on a microscope slide. We compared bias and precision for photosynthetic parameters estimated through conventional PAM fluorometry on intact biofilms and through our novel slide-based technique, both theoretically and empirically. Numerical simulations confirmed the consistent overestimation of key parameters for intact biofilms, with relative errors up to 145%, compared to, at most, 52% on thin films. Paired empirical observations likewise demonstrated that estimates based on intact biofilms were consistently higher (up to 248%, p<0.001) than estimates from thin films. Numerical simulation suggested greater precision with the slide-based technique for homogeneous biofilms, but potentially less precision for heterogeneous biofilms with improper subsampling. Our empirical comparison, however, demonstrated some improvement in precision with the slide-based technique (e.g., the coefficient of variation for the maximum electron transport rate was reduced 30%, p=0.009). We recommend the use of the slide-based technique, particularly for biofilms that are thick or have small light attenuation coefficients. Care should be taken, however, to obtain vertically representative subsamples of the biofilm for measurement.

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