FeMO3 Dive Cruise 2008
Report -- How Many Microbes Fit in a Mat?

Jason retrieves a "Bacterial Trap" with abundant
"Mat" material on top of it. But how many microbes
would fit in this mat?

Just about every day on this cruise I spend a little time in the Jason control van helping decide which experiments are deployed or collected from Loihi. The other day I was watching Jason retrieving an incubation that was intended to “trap microbes” so we can identify the microorganisms living at Loihi. Looking at the mat on top of the experiment some of the scientist began to contemplate the number of microbes in the mat that had formed on the top. We estimated there are more microbes in the mat on top of the microbe trap, than people living in the United States. So, at Loihi, if we assume that 0.01% of the seamount is covered with mat, has a surface area of 78 km2 and 1x108 microbes per cm2, there are approximately 10,000,000,000,000,000 microbes (1x1016) at Loihi! That is 10,000,000 (107) times more microbes at Loihi than people on this Earth (7x109)

A Petri dish with iron, baking soda
and vitamins dissolved in seawater
for growing Fe-loving microbes.

How to Isolate and Identify Microorganisms from Loihi?

Because the microbes are so small, even under the microscope, it is difficult to distinguish between microbes and minerals (the rocky materials where they are living on/from at Loihi). However, I can add a stain that that selectively binds to DNA of the microorganisms present I can recognize the microorganisms. But what kinds of organisms are living at Loihi? I can’t tell which microorganisms are there just by looking under the microscope. I do see unique iron structures, produced by iron-oxidizing bacteria (also referred to as iron-bacteria or iron-spinning bacteria) suggesting some of the cells are iron-oxidizing bacteria.

Traces of microbes on volcanic glass are very hard to distinguish just with
polarizing light microscope. Instead we need some more sophisticated
techniques to make them visible to us.

I also wonder “If I were a microorganism living at Loihi what would I eat?” While we eat complex foods like spaghetti, salad, cereal and vitamins, microbes eat compounds like glucose (sugar) or acetic acid (vinegar) and have the enzymes to make their own vitamins. Microbes can also use compounds that we find unappetizing such as iron, sulfide (the smell of rotten eggs) or methane (the gas from cows). Loihi waters contain high concentrations of iron, low concentrations of oxygen, and some carbon dioxide. To grow the microbes from Loihi in our laboratory I thus add iron, vitamins (just to give them a boost) and baking soda to seawater in a Petri dish. Then I add some of the mat and put the plates in a jar with a low concentration of oxygen. Even when I look at my cultures after a several days, I already see some of the stalk-producing bacteria. I think the stalk-producing organisms are related to Mariprofundus ferrooxydans, an iron-oxidizing bacterium previously isolated from Loihi, but I will have to purify the bacteria to be sure.

What About the Other Microbes we See? Who are They?

Unfortunately, not all the microorganisms present in mat are growing with the food I provided for them and I am still wondering, “Who are those microbes under the microscope?”

Fe-oxidizing microbes Mariprofundus ferrooxydans forming Y-shaped stalks.

All organisms contain DNA sequences that code for universal proteins, such as replication or protein production. Some of the scientists on this cruise previously sequenced the DNA from Mariprofundus ferrooxydans and the Loihi mats. These DNA sequences are similar but have a slight variability. The sequence variations can be used to generate a phylogenic tree (like a family tree) and describe the relationship of the organisms in the mats, Mariprofundus ferrooxydans and other known organisms to one another.

Petri dishes stored in an
airtight gas pack.

But how can I use this family tree to tell me who is under the microscope? One of the scientists on this cruise designed a molecular probe that has a fluorescent molecule on the end of a short (but unique) sequence of DNA that matches the DNA of Mariprofundus ferrooxydans and it’s cousins. I can apply this probe to samples of mat, identify the microorganisms present and see which cells are attached to the iron structures. Here is what happened when I applied a molecular probe to with Mariprofundus ferrooxydans in the lab.

DNA staining enlightening the existence of living microbes at the end of each
Y-shaped Fe-oxide stalk. Now we finally can count how many microbes live
in the mats at Loihi seamount.

Viola! The probe only lights up the cells that contain the sequence of the probe and I know who is who under the microscope. Notice how the cells are attached to the iron structures. When I return to the lab back in Maine I will use molecular probes to determine the percentage cells in the mat that are iron-oxidizing bacteria. If only a 10th of the microbes there are iron-oxidizing bacteria, that would still be 1015 microbes at Loihi that love to have iron on the menu!

Emily Fleming onboard the R/V Thomas G. Thompson
8 October, 2008

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