FridayAFM - the bacteria in the shell

Héctor here, your AFM expert at Nanosurf calling out for people to share their Friday afternoon experiments. Today sand reveals the smallest cave dwellers ever known.You will learn:

• More about seashells.
• Sample preparation.
  

Last week I travelled North, as far north as I have ever been. Let's put it like this, there were no nights, I was under the 24h sun.

I visited Trondheim in Norway to train PhD students belonging to the TOPOCOM network and deliver a workshop on AFM during the ECAPD conference.

(Yes, there was an AFM doing live imaging on the stage).

During the conference there was very little time to prepare fridayAFM... so there was almost no post this week. Almost. But what actually happened is that I stumbled upon a research topic that has been quite active in recent years.

During the weekend I visited the Munkholmen isle (so tiny that I think the right term is islet), and while sunbathing on the beach, I realized the sand was made, among other things, of broken seashells.

Seashells have been the topic of fridayAFM several times (most recently on FridayAFM - Black cone shell periostracum which added new data and summarized previous results), so there isn't too much novelty on imaging seashells again. In fact, the best I could came up with was "maybe these have smaller aragonite tablets, or with different shapes...", but I couldn't be more wrong, and in fact what I saw could help clarifying a few open questions in maritime biology.

With several samples from the beach, I returned to the conference, and in between sessions showcasing the capabilities of the DriveAFM, I prepared the samples for AFM imaging. In this case, since the sand was quite dry and it left few residues (or at least not many that I could see with the naked eye), I simply glued the samples to a piece of metal without further cleaning.

As a glue I used a trick from my colleague Amelie Axt. Clockmakers goo that fixes small pieces without residue.

Then I started imaging in Dynamic mode with Tap 190 probes.

C is the easiest to identify as a shell. No big surprise here, and interestingly, the shapes and sizes of the aragonite tablets are similar to the ones on the mother pearl. E is clearly a shell (to he naked eye), so most likely the aragonite tablets have been eroded and what we see is the tablets arranged periodically, but with the borders smoothed. A in the other hand is not so clear. It could be what happens under extreme erosion or it could be a stone. From the shape and feel to the naked eye I would say it is a shell, but could easily not be.

What about the others? The ones with stripes are below.

They are both stones. E shows no big differences between the stripes, because the dark areas have been smooth down. B on the other hand, shows big differences, and the bright areas are quite nice because they show two types of structures, big rounded shapes, and smaller grains underneath.

So.... ok, interesting.... but where is the promised interesting result?

It is in sample C, the shell with well defined borders. At first glance it doesn't look like much... but upon further inspection...

One notices that is is filled with lots of holes everywhere. About 200 nm in diameter. This is something new which we didn't saw in any of the previous fridayAFMs on seashells.

What are these holes?

It turns out these holes are likely due to Cyanobacteria digesting the material of the aragonite tablets in a complex process involving light, Ca ions, and CO₂ (See refs 1 to 4). However, It is not clear if the tablets grow around the bacteria and when it dies it leaves a cave, or the bacteria bores onto the tablets making the holes as it goes deeper onto the aragonite tablet.

To my knowledge, these are the first AFM images of such holes. Maybe the next time we image in liquid and manage to see the bacteria poking out of the holes?

Let's recap. What seemed as a boring AFM sample turned to be a bacteria boring sample. Little holes on the aragonite tablets forming seashells led to an interesting research topic, that of bacteria liberating CO₂ from calcite deposits. Can these bacteria eventually liberate all the CO2 absorbed in calcite and how much they contribute to the global warning effect? Do they affect the animal when alive or their effect on the living quality of the host is minimal?

I hope you find this useful, entertaining, and try it yourselves. Please let me know if you use some of this, and as usual, if you have suggestions or requests, don't hesitate to contact me.

References:

[1] Wu G, Huang A, Wen Y, Wang H, Wang J, Luo F, Wu M. Euendolithic Cyanobacteria and Proteobacteria Together Contribute to Trigger Bioerosion in Aquatic Environments. Front Microbiol. 2022 Jul 6;13:938359. https://doi.org/10.3389%2Ffmicb.2022.938359 

[2] Lawfield, A. M. W., Gingras, M. K., & Pemberton, S. G. (2014). Microboring in a Freshwater Fluvial Unionid Bivalve Substrate. Ichnos, 21(3), 193–204. https://doi.org/10.1080/10420940.2014.934190 

[3] Tietze, Eleonor and Karina Soledad Esquius. “First record of cyanobacteria microboring activity in Pampean shallow lakes of Argentina.” REVISTA BRASILEIRA DE PALEONTOLOGIA (2018): n. pag. https://doi.org/10.4072/RBP.2018.2.08 

[4] Ćurin, M., Peharda, M., Calcinai, B., & Golubić, S. (2013). Incidence of damaging endolith infestation of the edible mytilid bivalve Modiolus barbatus. Marine Biology Research, 10(2), 179–189. https://doi.org/10.1080/17451000.2013.814793