In vivo growth fluorometry: accuracy and limits of microalgal growth rate measurements in ecophysiological investigations

Abstract

ABSTRACT:In vivo growth fluorometry (Karsten et al. 1996) is based on the sensitive detection of in vivo chlorophyll a (chl a) fluorescence and monitors its increase over time as an indicator for growth. The method offers a simple, rapid, non-invasive, reproducible and calibration-free measurement of growth rates in unialgal cultures and facilitates multifactorial ecophysiological studies on algal cultures. The technical setup is well suited for use on benthic, adhering, filamentous and colonyforming microalgae. Low detection limits avoid self-shading and nutrient limitation during growth rate determination. Acclimated growth rates should be measured after a pre-incubation phase of 1 to 3 d. A broad data set correlating in vivo chl a fluorescence with cell number, organic carbon and chl a concentration in representative members of the Cyanobacteria, Rhodophyta, Chlorophyta and Bacillariophyceae is presented. A calibration of chl a fluorescence is not required for acclimated growth rate measurements of Bacillariophyceae and Chlorophyta but is recommended for high abundances of Cyanobacteria and Rhodophyta.KEY WORDS: Growth rate · Chlorophyll a fluorescence · Adhering algae · Multifactorial ecophysiological studies Resale or republication not permitted without written consent of the publisherAquat Microb Ecol 55: [95][96][97][98][99][100][101][102][103][104] 2009 ical) determination of abundance is often replaced by cellular biomass markers, such as chlorophyll a (chl a), carbon, ATP or proteins (Stein 1973, Butterwick et al. 1982, Becker 1994. Disadvantages of using these biomarkers are related to incomplete extraction procedures, calibration protocols and the specificity of the test methods (for discussion see Wright et al. 1997). Moreover, the absolute concentration of biomass parameters is influenced by abiotic parameters such as radiation or nutrient supply, and by culture age, physiology and size of cells (Kruskopf & Flynn 2006). In laboratory cultures these factors can be controlled, while respective data obtained in field studies must be evaluated by taking into account alterations in community structure and variation in abiotic parameters. Furthermore, the use of carbon or protein content as algal biomass equivalents is hampered by co-occurring bacteria or organic exudates.Growth of colonial or filamentous organisms can be determined gravimetrically as dry mass (e.g. GarciaPichel et al. 1992). Packed cell volume measurements offer a similar approach, as algal samples are centrifuged in scaled capillary tubes and their abundance is determined as biomass by volume. However, dry mass and packed cell volume include any contamination such as bacteria or suspended particles and, hence, falsify the results.The most widespread in vivo method in culture studies is the determination of absorbance (optical density) at a given wavelength. It is a simple and non-invasive method for measuring microbial abundance. Optical density is commonly defined as log 10 (I 0 /I t ), i.e. the logarithm to the base 10 of the ra...

Investigation of the algicidal exudate produced by Shewanella sp. IRI-160 and its effect on dinoflagellates

“…Fluorescence has been shown in other studies to be a good proxy for algal biomass under nutrient replete/logarithmic growth conditions (e.g. Brand et al, 1981;Brand, 1985;Doblin et al, 1999;Gustavs et al, 2009;Maldonado and Price, 2001;Wood et al, 2005). All experiments were conducted using logarithmic cells except for the growth phase experiments where cell counts were performed to ensure that fluorescence was an accurate measure of algal abundance.…”

Ecophysiological performance of an urban strain of the aeroterrestrial green algaKlebsormidiumsp. (Klebsormidiales, Klebsormidiophyceae)

“…Growth responses under a range of photon fluence densities and salinities were followed using an in vivo growth fluorometer (Hansatech MFMS, Norfolk, UK) according to the methodological approach of Gustavs et al (2009b), which monitors increases of in vivo chlorophyll a fluorescence F t over time as an indicator for biomass accumulation (Karsten et al, 1996). Fluorescence measurements were performed every 24 h for up to 10 days.…”

“…All measurements started with the addition of 150-200 ml log-phase stock culture. Growth rate was calculated as the mean value from replicate samples using the equation F t ¼ F 0 e mt (F 0 : initial fluorescence; F t : fluorescence after t days) for each individual sample (Gustavs et al, 2009b).…”

“…did not grow under these hypersaline conditions, it survived 45 psu while at 60 psu many filaments became bleached (data not shown). In vivo growth was measured using a fluorometer according to the technique of Gustavs et al (2009b). Significance of differences among the treatments was calculated by one-way ANOVA (P < 0.001).…”

Comparing the diel vertical migration of Karlodinium veneficum (dinophyceae) and Chattonella subsalsa (Raphidophyceae): PSII photochemistry, circadian control, and carbon assimilation

“…In-vivo chlorophyll-a fluorescence of dark acclimated samples was used as a proxy of cellular biomass [23,101,33]. Use of this proxy provided an efficient way to measure algal distribution, and facilitated high-resolution spatio-temporal sampling strategies.…”