PMC1360661_01
Maximum intensity images (A, B, C) and vertical profiles of biofilm coverage analyzed by PHLIP (D, E, F) of a biofilm grown on glass slides in natural seawater (Oosterschelde, the Netherlands). Images and vertical profiles are shown for representative samples measured at days 3 (A, D), day 25 (B, E) and day 44 (C, F). The red channel and open circles represent bacteria stained with syto64, the green channel and black squares represent EPS labeled with ConA and the blue channel and crosses show chlorophyll a autofluorescence. CLSM maximum intensity images represent areas of 250 m x 250 m.
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F1
A selection of maximum intensity projection images and the accompanying vertical profiles from image stacks recorded at different time points during the experiment are shown in Fig. 1. After a period of 3 days (Fig. 1A), biofilms were dominated by bacteria that were partly embedded in ConA-labeled EPS of the biofilm matrix. Changes in the morphology of the chloroplasts indicated a succession in the algal community with a general shift towards larger species (Fig. 1B,C). The thickness of the biofilms increased from about 30 m after 3 days of incubation (Fig. 1D) to 200 m in 44-day-old biofilms (Fig. 1F). Bacteria were only observed as a significant fraction in the 3-day-old biofilm where they were present near the substratum (Fig. 1D). Part of the algal community was present close to the substratum while the remaining part extended into the biofilm (Fig. 1E,F). This is in agreement with the changes in morphology of the chloroplasts as observed in the CLSM images (Fig. 1), indicating a succession of diatoms over time from small species to larger species.
Temporal development of a natural phototrophic biofilm grown on glass slides in natural seawater (Oosterschelde, the Netherlands). All-channel image analyses of (A) biovolume, (B) average thickness and (C) roughness. Values indicate mean +- S.D., (n = 21).
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F2
This led to a significant increase in biovolume, thickness and roughness of the biofilm (Fig. 2A,B,C). All-channel analyses resulted in a significant linear increase in biovolume (Fig. 2A, R2 = 0.9704, p < 0.001). Biofilm thickness (Fig. 2B) also increased over time (p < 0.01) but the temporal evolution was best predicted by an exponential decay function (R2 = 0.8855, y = 0.33-0.29xe(-x/16647)). Even though a linear correlation between roughness and days of incubation (Fig. 2C, R2 = 0.9328) was observed, statistical analysis showed that this trend was not significant (p = 0.57).
Temporal development of a natural phototrophic biofilms grown on glass slides in natural seawater (Oosterschelde, the Netherlands). Image analyses of separate channels representing chlorophyll a fluorescence (black bars), EPS stained with ConA (open bars) and bacteria stained with syto64 (dashed bars). (A) biovolume and (B) average thickness. Values indicate mean +- S.D., (n = 21).
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F3
Analysis of the information from different channels acquired by CLSM revealed that different biological components in the biofilms (i.e. bacteria, micro-algae and the ConA-stained EPS) followed different dynamics in time (Fig. 3, 4). In contrast, a steady increase (p < 0.001) was observed for the chlorophyll a biovolume (Fig. 3A). Average thickness of the biofilm, as determined from each individual component, increased over time for all channels (p < 0.001 for chlorophyll a and ConA, p < 0.05 for syto64), but thickness of ConA was significantly higher compared to the chlorophyll a and syto64 signals (p < 0.001 and p < 0.05, Fig. 3B).
Development of morphological parameters analyzed from a phototrophic biofilm grown on glass in natural seawater (Oosterschelde, the Netherlands). (A) Temporal development of area-to-volume ratio. Separate channels represent chlorophyll a fluorescence (black bars), EPS stained with Con A (open bars) and bacteria stained with syto64 (dashed bars). Values indicate mean +- S.D, (n = 21). (B) The correlation between biofilm roughness and biovolume. Filled squares: chlorophyll a fluorescence, open circles: ConA-fluorescence, open triangles: syto64, filled triangles, all channel signal.
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F4
Analysis of the information from different channels acquired by CLSM revealed that different biological components in the biofilms (i.e. bacteria, micro-algae and the ConA-stained EPS) followed different dynamics in time (Fig. 3, 4). Variations in the morphological parameters area-to-volume ratio and biofilm roughness occurred over time (Fig. 4). Area-to-volume ratios of chlorophyll a showed a steady decrease after 5 days of incubation (p < 0.001, Fig. 4A). When the estimates of biofilm roughness were plotted as a function of biovolume, a correlation was found between the parameters (Fig. 4B).
Maximum projection images of a biofilm grown on glass slides after incubation in natural seawater (Oosterschelde, the Netherlands). Visualization of bacteria, EPS and chlorophyll a (A) and reflection image (B) of a 31 day old biofilm. The red channel represents bacteria stained with syto64, the green channel represents EPS labeled with ConA and the blue channel shows chlorophyll a autofluorescence. In addition, the depth profiles of biofilm coverage for ConA (filled squares), syto64 (open circles) and chlorophyll a (crosses) are given (C) as well as the depth profile for the reflection signal (D). The CLSM images represent areas of 250 m x 250 m. White arrows indicate the different reflective structures containing bare glass substratum (I), silica frustules of diatoms (II) and amorphous material (III).
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F5
In addition to the quantification of biological components, which included bacteria, micro-algae and EPS, reflection images of biofilms visualize solid inorganic material (Fig. 5) and can be similarly analyzed using PHLIP as illustrated here. Biovolume estimates showed that for the image stack in Fig. 5, about 85% of the biofilm constituted of inorganic reflective material, while organic material represented the remaining 15% of the total biovolume.