A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated (WATER-PAM) Fluorometry

Abstract

Conventional methods for experimental manipulation of microalgae have employed large volumes of culture (20 ml to 5 L), so that the culture can be subsampled throughout the experiment1-7. Subsampling of large volumes can be problematic for several reasons: 1) it causes variation in the total volume and the surface area:volume ratio of the culture during the experiment; 2) pseudo-replication (i.e., replicate samples from the same treatment flask8) is often employed rather than true replicates (i.e., sampling from replicate treatments); 3) the duration of the experiment is limited by the total volume; and 4) axenic cultures or the usual bacterial microbiota are difficult to maintain during long-term experiments as contamination commonly occurs during subsampling. The use of microtiter plates enables 1 ml culture volumes to be used for each replicate, with up to 48 separate treatments within a 12.65x8.5x2.2 cm plate, thereby decreasing the experimental volume and allowing for extensive replication without subsampling any treatment. Additionally, this technique can be modified to fit a variety of experimental formats including: bacterial-algal co-cultures, algal physiology tests, and toxin screening9-11. Individual wells with an alga, bacterium and/or co-cultures can be sampled for numerous laboratory procedures including, but not limited to: WATER-Pulse-Amplitude-Modulated (WATER-PAM) fluorometry, microscopy, bacterial colony forming unit (cfu) counts and flow cytometry. The combination of the microtiter plate format and WATER-PAM fluorometry allows for multiple rapid measurements of photochemical yield and other photochemical parameters with low variability between samples, high reproducibility and avoids the many pitfalls of subsampling a carboy or conical flask over the course of an experiment.

Niche-based assembly of bacterial consortia on the diatom Thalassiosira rotula is stable and reproducible

“…In growth experiments, diatom growth was monitored daily in black 96-well polystyrene microplates with clear bottom (Greiner Bio-One, Germany) by measurements of relative fluorescence units (RFU) of chlorophyll using a plate reader (FLUOstar Omega, BMG, Germany) at optical filters settings of λ ex 440-80 nm and λ em 640-80 nm. In Experiment I diatom growth and performance were further monitored by pulsed-amplitude-modulation fluorometry (Water-PAM, Walz, Germany) by measurement of the minimal (F 0 ) and maximal (F m ) dark fluorescence [35]. Briefly, samples were taken 5-7 h into the light cycle and diluted in ESAW to be within the PAM detection range.…”

The Bacterial Symbiont Phaeobacter inhibens Shapes the Life History of Its Algal Host Emiliania huxleyi

“…When grown alone, all three algal cell types had a stable PSII maximum quantum efficiency (F v /F m > 0.5) throughout the experiment, although they entered a senescent stage, represented by reduced fluorescent health after 6-10 days (Figures 2A-C). The algal cultures grown alone did not die in this experiment and have been shown to live for > 60 days in microtitre plates (Bramucci et al, 2015). Both C and S cultures grown alone reached their peak density between 6 and 10 days followed by a characteristic slow decline during senescence (Figures 3B,C), typified by gradual losses of cell numbers (Franklin et al, 2012).…”

“…Bacterial-algal co-cultivation was performed as previously described (Bramucci et al, 2015). Briefly, stationary phase bacterial cultures were washed twice by centrifugation and resuspended in L1-Si medium before further centrifugation and resuspension to the target cell concentration in colony forming units (CFU)·mL −1 .…”

“…A PAM fluorometer (WATER-PAM, Heinz Walz) was used to measure chlorophyll fluorescence (Schreiber et al, 1986;Bramucci et al, 2015). All samples were taken at the midpoint of the dark cycle (4 h) and diluted in sterile L1-Si medium to within the detection range of the PAM fluorometer.…”

First insight on interactions between bacteria and the marine diatom Haslea ostrearia: Algal growth and metabolomic fingerprinting

“…Azospirillum brasilense, a plant growth-promoting rhizobacteria (PGPR), known for its ability to stimulate the growth of terrestrial plants and microalgae [36], was included in the set of bacteria to be tested in co-culture with H. ostrearia as a potential positive control. [37]. This non-invasive method, based on chlorophyll fluorescence for measuring algal growth of multiple parallel cultures, has already been used by Le Chevanton et al [7] and Zecher et al [38] for the co-culture of the bacterium Rheinheimera sp.…”