It was a cold rainy post-laboratory afternoon when my good friend Maria, one of the founders of Entire Ocean, invited me to write an article for her website. I was flattered with the invitation but, what could I write about that would honor the beautiful and dedicated work made by Maria and Aja in Entire Ocean? Since at that time they were both developing their Master thesis on the same laboratory where I was working, and we were frequently helping each other, I couldn’t help but thinking on an article that describes perhaps the most common task performed on research laboratories around the world: problem solving.

As a follower of a website called Entire Ocean, you probably already know that the ocean has, on average, a salinity of 35 S (the S stands for an unnecessary unit of salinity, since it is the same as ‰ or grams per liter). This means that if you fill a one-liter bottle with seawater and leave it somewhere for a sufficient time so that all the water evaporates, approximately 35 grams of salt will remain in the bottle. That is quite a lot! Most of this salt will be sodium chloride (NaCl), a combination of two of the most abundant ions in solution in seawater (Na⁺ and Cl^–) and the salt that we use to season our food. In the remaining material within your bottle, there will also exist dry biomass, coming from marine living organisms (yes, even if you didn’t catch a fish or a shrimp, there will be biomass).

The marine environment is replete with microorganisms, invisible to the naked eye. In fact, a glass of seawater may contain a microbial biodiversity comparable to entire terrestrial ecosystems – of course, not any terrestrial ecosystem. I do not know the average microbial biomass dry weight in seawater. If such data exists, it will certainly be overwhelmingly variable depending on seasonal and spatial factors. However, considering that, in laboratory, dense cultures of marine phytoplankton yields circa 1 gram of dry biomass per liter of growth medium, let’s exaggerate and use this as a reference value. Finally, then, in our waterless bottle we will find 35 grams of salt plus 1 gram of dry marine microbial biomass. In case you still did not realize, this is a problem, a salty problem!

The research of marine microorganisms relies on several procedures in which the correct determination of biomass dry weight is required. Not only this value per se is important, but also it is determinant for gravimetric analyses (those where the weight of an extract versus the weight of biomass gives you the extract concentration).

But how can scientists accurately determine something as insignificant as the microbial dry weight within that much higher salt content? Can’t they just wash it with freshwater? Can’t they just make an approximation?

Early in my marine-scientist career, these questions made me think on how thoughtful science really is. If an information as trivial as “seawater contains salt” drives to that many considerations, I could only imagine the premises based on less obvious characteristics of seawater.

Nevertheless, the fact is that in laboratories microorganisms are not harvested by simple evaporation of seawater; cultures are usually centrifuged (a process where cultures are rotated fast enough so that the centrifugal force pulls microbes down, forming a uniform mass of cells called pellet). A great part of the salt is discarded in the cell-free remaining liquid (that can be seawater or growth medium) called supernatant. Unfortunately, the pellet is not dry enough, and the salty problem remains in a smaller but relevant scale. In fact, the bigger the cells, the bigger the problem, since the interspace between cells is directly proportional to the cell size (It gets more and more complex! Figure 1).

Figure 1: The result of a centrifugation: supernatant is discarded with a good amount of salt, but the pellet retains water. As you can see in the details, the bigger the cells the more salty water is retained and, hence, salt weight compromises more the measurements.

The issue of washing marine microbes with freshwater is that marine organisms are physiologically adapted to the high concentrations of salt. Basically, it is the same problem as putting a freshwater aquarium goldfish in seawater or, the other way around, putting a marine clownfish in freshwater. Very few species can tolerate this stress. The solution is to wash pellets with another salty water, having the same concentration of seawater but a different salt. The new salt is volatile at mild temperatures and, as the biomass dries on an oven, the salt decomposes into gas (those are usually ammonium salts) leaving only the microbial biomass to be analyzed.

It is so beautiful to see the careful attention that science gives to the simplest aspects of its materials and methods. And it is thrilling to know that most of these simple problems are still humbly open for investigation. Our salty problem, for instance, is far from being consensually solved. There are several different protocols, using different salt solutions, with different steps and ways of washing the different microbes. The simple act of choosing a protocol implies on a considerable amount of studying time. In my opinion, that is the marine analogy to the reason why people should trust science and value scientists. Regardless of my (our) love for the oceans, that is a message I am always happy to pass on!

I hope to have fulfilled the expectations of Entire Ocean,

Daniel Kurpan, PhD.

P.s. Stay tuned for penguins! 🙂