Science on a Ship: Diverse Microbes of the Deep, Dark Oceans

How did you develop an interest in studying microbes below the seafloor?
The field originally started this way with the study of things at the level of the seafloor. Later, at hydrothermal vents where fluids come out of the seafloor, it was observed that these fluids contained microbes. People wondered where these microbes came from and speculated that it’s likely below the seafloor. And so, there came an interest in learning what microbial community lies below the seafloor.

Why is it important to study the oceanic microbes?

There are two major parts to seafloors: a top layer of sediments i.e. mud on the seafloor which is spread over the second, bottom layer of rocks which form a part of the Earth. I largely study rocks that make up the actual sea floor. There are different reasons for studying microbes that live in both layers.

Residual organic carbon from dead oceanic life in sunlit waters falls to the seafloor, a process known as carbon burial. This carbon can get deeply buried into the seafloor. Learning about the microbes that live on and below the ocean floor, that thrive on the ocean’s carbon burial, is really important for understanding global carbon cycling. And what’s fascinating to me about the subseafloor layer is that water is moving through those rocks below the sediments – in any one period of time, around 2% of the volume of the ocean is moving through this subsurface aquifer. So, it’s important for us to know what’s happening in that aquifer, both chemically and biologically, because it does have a big impact on the broader ocean chemistry.

When you’re at sea what’s your typical working day like?
It somewhat depends on the type of expedition. For the drilling type of expedition, where we methodically extract samples at different depths below the seafloor, it’s a bit more routine: there’s only one type of sampling, and so generally with those cruises, we’ll work shifts of 12-14 hours a day. A sample will come up every few hours, and we have a set of procedures to immediately preserve these samples – that’s a bit more regular.

With some of the other expeditions using robots and submarines, there are periods of quiet and then intense periods of work when samples actually come up – a lot of prep work, and then some waiting… and then the minute the samples come up, you’re working really hard for about 3-4 hours to keep microbes alive and analyze them.

Can you tell us about your lab’s projects?
My lab has two focuses: one aspect of what we do is trying to understand and characterize the diverse microbial community at and below the seafloor, so at places like hydrothermal vents which are at the seafloor and also below the seafloor in the rocks. The other half of my lab looks at the microbial ecology and its relation to how humans interact with the environment. We currently have a large project looking at microbial responses to oil spills. For this, we go out and collect a lot of water from surface oceans, store them in 100-liter tanks and then have experiments to analyze the microbes in them under different conditions.

Dr Sylvan at work aboard the JOIDES Resolution in January 2016, sampling rocks from below the seafloor to determine their enzyme activity rates Image credit: Virginia P. Edgcomb, WHOI

You have a functioning lab while on a cruise. Tell us about your on-board experiments.
When the samples come up from the drilling, it’s a great amount of work to capture the biochemistry of the microbes and keep them viable. On the ship, I largely use Tecan’s Spark microplate reader to look at microbial enzyme activity. We have fluorescence-based assays to study the rates of phosphatase activities in these microbes. The initial substrate is non-fluorescent, but with increased microbial enzyme activity, the phosphate gets cleaved and there is a fluorescent response. We can now measure fluorescence using the Spark plate reader that directly represents the activity of the microbes. I can analyze a number of samples, sometimes in duplicates and triplicates, to yield more accurate data, multiple times a day in a 96-well format. That’s pretty powerful.

And the oil spills project…?
For the oil spills project, we have large experiments that are completed on land, so I can bring the plate reader to the lab where the experiments occur. One of our questions is that after an oil spill in the ocean, what is the response to the microbial community – how is their survival and how quickly are they now growing? I look at enzymes related to carbon, nitrogen and phosphorus utilization. These are three crucial elements every microbe needs to grow. They can tell you about the overall nutritional needs of a microbial community. There are twelve 100-liter tanks with water from the ocean surface, with different treatments – control; with oil; and, oil plus a chemical used to clean up oil spills. And then we sample the microbial communities every 12 hours over a course of four–five days. We measure three different enzymes, one for carbon (beta glucosidase), another for nitrogen (leucine aminopeptidase) and phosphorus (alkaline phosphatase). These are present at the cell surface or in the periplasm so they are related to the cell’s response to the outer environment, and not necessarily to reactions inside the cell. We use a similar fluorescence-based assay for the measurement of these microbial activities on the plate reader as well.

When you’re mid-ocean, you may not have the same experimental liberties as scientists that are on land. How do you decide what comes with you on a cruise?
We generate a lot of samples from very rare, faraway locations. Having a multimode microplate reader like the Spark on-board not only lets me measure the activity of these microbes but also provides kinetic curves. This instantly helps determine the saturation of the substrate to ensure, in real-time, that you’re measuring in the right concentration range. If I couldn’t do all this with my samples at once, it would take me days, whereas now I can do it within a matter of hours.

What’s the future of your research?
At its core in our lab, we’ll always remain interested in understanding how different environments select for microbial community members and, in turn, how these microbial members influence the environment around them. There will be a fair amount of molecular biology but at the end of the day, I’m still interested in rates of microbial processes – I want to know how quickly these things are growing, how they affect the environment chemically. For that, it’s always going to come back to physiological measurements that are largely made with microplate readers.

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