Movement in EPS matrix

Biofilm of Craticula cuspidata (30x time lapse) Pinnularia viridiformis within the biofilm and on its surface (25x timelapse)

 

Movement of diatoms in and on biofilms

According to the explanations for the active motion of pennate diatoms one expects that a direct or indirect contact between raphe and a substrate is necessary. The requirements on the hardness of the substrate are often not high, because movements on agar are possible (Iwasa, K., Shimizu, A., 1972).

Motile pennate diatoms that form biofilms, like many diatoms of the genus Pinnularia, Rhopalodia or Craticula can move well in their biofilm. A biofilm that has detached itself from the substrate is shown on the page about nutrient solution. In the top left video such a detached biofilm of Craticula cuspidata (Length: approx. 120 µm) can be seen under the stereomicroscope in dark field illumination. One can recognize a strong movement of the diatoms in the transparent biofilm.

Biofilm in Pinnularia viridiformis cultures

Diatoms of genus Pinnularia excrete extracellular polymeric substances (EPS) during the activity of the raphes, which by and by can form a soft deposit in the cultures above the bottom of the petri dish (overview of EPS in diatoms in Aumeier, C. and D. Menzel, 2012). On the left a very small Pinnularia (length approx. 87 µm) is to be seen, which very quickly builds up a thick biofilm (click to enlarge). It is a Pinnularia viridiformis morphotype 3 sensu Krammer 2000.

At the beginning the video on the upper right shows three diatoms of this species which are staying at the bottom of a petri dish. On top of that a layer of EPS is deposited, on which most diatoms are located. Afterwards these diatoms are brought into focus. Movements can be observed in both layers with movement on the biofilm predominantly occurring in pivoting motion. At the bottom of the biofilm the Pinnularia viridiformis often straighten up vertically, which they do not do in pure nutrient solution.

Under the stereomicroscope diatoms are easily found between the bottom of the petri dish and the surface of the biofilm. The diatoms between the bottom of the Petri dish and the surface of the film, which are rare compared to the population on the surface, often rotate around changing axes. The video on the left shows Pinnularia viridiformis within the biofilm. The time-lapse factor is 12 times as large as the one in the video at the top right. In the background, the faster moving diatoms on top of the biofilm can be seen out of focus. I assume that a migration of the diatoms from the bottom to the top of the film is possible. To the opposite direction such a change is presumably rare or impossible. This is suggested by the found far greater number of diatoms on top of the biofilm.

Properties of the biofilm

The layer of EPS shows viscoelasticity, so it is neither purely viscous nor purely elastic. The viscous property of the gel allows the movement of the diatoms at the bottom of the petri dish and within the biofilm. Because of the elasticity the diatoms are able to stay on the surface without sinking to the bottom of the petri dish.

As the biofilm is colorless and transparent, its surface cannot be recognized under the microscope. If small particles are placed from above, these remain on the gel and mark the surface of the biofilm to the water.

In the observed culture (petri dish with diameter of 5.5 cm) the thickness of the biofilm was between about 150 µm and 380 µm. For comparison, the length of this Pinularia is at about 90 µm. At the time of recording the culture was 30 days old. Locally the layer is flat enough to focus the diatoms in the field of view at the same time.

Use of the biofilm to study the activity of the raphes of Pinnularia

The elastic properties in connection with the possibility of marking the boundary surface between the gel layer and the water can be used to observe the activity of the raphes. The modulus of elasticity proves to be so small that elastic deformations which are easily observable occur during the movement of the diatoms. In the experiment mineral particles with a linear expansion of typically less than 3 µm were distributed on the gel layer. In the video on the left (30x time lapse) one can see how the surface of the gel layer is elastically deformed by the movement of the Pinnularia viridiformis. It is striking that there is a slow build-up of tensions and a rapid, jerky drop of the tensions. The cause for this becomes understandable if one considers the process at a location with a low population density. In case of steady movement of the diatoms no conspicuous deformations occur. Frequently however, the movement of the diatoms stops without the raphes ending their activity. Through the transparent valves one can see that both raphes are now working towards their proximal ends. The gel in the vicinity of the diatoms is thereby elastically deformed due to the adhesion of the raphe to the substrate. It is compressed (negative strain) at the proximal ends and in the neighborhood of the diatoms in the direction of the transapical axis. A short video of such a process together with a superimposition of the frames during the standstill of the diatom can be seen below (click to enlarge):

The deformation has a visible effect at a distance of several diatom lengths.

After a while the raphes no longer work against each other, and the movement is continued in the same or opposite direction. The EPS layer returns to its equilibrium position. The gel expands in a short time, which is reflected in the movement of the gel surface in some distance from the diatom.

Pinnularia viridiformis lie on a planar, solid substrate flat on their valves, as can be seen from the silhouette of the valves in the belt view. It can be assumed that raphe systems that work against each other also in this case cause the movement to stop.

In this type of motion the diatoms consumes energy. It would be interesting to know whether this energy consumption is useful, or whether there is a lack in the coordination of the raphes. This question also arises for another reason. It is easy to show in diatoms of the genus Pinnularia as well as in many other diatoms that often raphes are active which are not coupled to the substrate and thus do not contribute to the movement.

A simultaneous activity directed to the distal ends of the raphe is not observed in this and several other videos. It would lead to an elastic expansion of the gel. With this observation technique it cannot be determined whether both raphe systems are involved in uniform motion of the diatom.

Apparently particles can also be displaced during the opposing activity of the raphe. In the upper video on the left, the initially evenly dispersed particles already show striking clusters.

The method provides in principle the possibility of quantitatively determining the forces exerted on the substrate by the raphe. For this purpose the elasticity module and the displacement of the particles have to be determined.

It is also a good idea to use this method to investigate the activity of the raphes even in species which do not produce such a biofilm. For this purpose a gel (agar or gelatin) could be superimposed with nutrient solution and the top of the gel layer could be marked with particles. The difficulty consists in the production of an elastic layer with a very small modulus of elasticity. If this fails, diatoms could be placed on the biofilm produced by Pinnularia.

 

Aumeier, C. and D. Menzel, Secretion in the Diatoms, in Secretions and Exudates in Biological Systems, J.M. Vivanco and F. Baluška, Editors. 2012, Springer Berlin Heidelberg. p. 221-250.

Iwasa, K., Shimizu, A., 1972. Motility of the diatom, Phaeodactylum tricornutum. Exp. Cell Res. 74, 552–558.

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