Research Feature: Studying microbes 450 meters underground

Link to the Full Article

M. E. Nielsen, M. R. Fisk, J. D. Istok, and K. Pedersen, 2006, Microbial nitrate respiration of lactate at in situ conditions in ground water from a granitic aquifer situated 450 m underground, Geobiology, volume 4, pages 43–52.

Posted: December 10, 2006

In March 2006, Mark Nielsen, a Subsurface Biosphere IGERT doctoral student, and his faculty advisors, published an article in Geobiology about microbial experiments they carried out 450 meters below ground in the Äspö Hard Rock Laboratory (HRL) in Sweden. The following interview gives a “behind the scenes” look at Mark’s research and its significance, both as a contribution to subsurface science and as an important learning opportunity for Mark.

What was the focus of your research project?

I was trying to measure the activity of microbes that live in a granite aquifer in the Earth’s deep subsurface.  The unique thing about the project was the setting – I worked 450 meters underground in the Äspö Hard Rock Laboratory (HRL) in Sweden so I was able to study the microbes at in situ conditions. 

Normally to research the deep subsurface biosphere, scientists have to work with drill rigs and make measurements remotely or remove samples for study in the lab.  But in a surface lab setting, it is hard to recreate the whole package of environmental conditions that the microbes experience underground – for example, it’s difficult to create a flow system with native groundwater and the right pressure and temperature conditions.  In the Äspö Tunnel I could study microbial activity while the organisms were still in their native setting.

Photo of the Aspo Tunnel.

Photo looking down the tunnel road. Note the steep 14% grade indicated in the warning sign to the right.

What was it like working in the Äspö Tunnel?

I like to compare it to the Petronus Towers - at 450 meters, they are the second tallest buildings in the world.  The Äspö tunnel is the equivalent depth but it goes in the opposite direction.  You can travel down into it either by road, one several kilometers long and big enough for tour busses to travel on, or by an elevator.  It was really humbling working there – when I went to collect samples on Sundays when very few other people were around, I felt so isolated.  I felt like the loneliest ant and if I turned off the lights it was totally pitch black – beyond the kind of dark of night. 

The lab is on the East Coast of Sweden and is used for many different kinds of experiments related to the deep burial of radioactive wastes.  There were multiple projects in alcoves along the tunnel with signboards about what was being studied.  The lab I worked in was inside a transport container with lab equipment and even internet access. 

Photo of the outside of the lab. Photo of Mark working inside the lab.
Photos of the outside and inside of the groundwater lab which was housed in a shipping container.

Why is the activity of subsurface microbes important?

In 1998 there was an important paper published by Barny Whitman and colleagues at the University of Georgia where he estimated the number of microbes living on Earth, including in the subsurface.  Everywhere below a certain temperature, microbes live in micro-fractures and pore spaces in sediment and rock and when he quantified how many microbes are out there, he calculated that there is probably more biomass in the subsurface than in any other part of the biosphere.  This means that if the microbes in these environments are biologically active, they could be ecologically very important.  For example, they could influence the chemistry of the atmosphere and the oceans.  Yet, microbes in the deep subsurface have been studied very little, especially microbes that live in crystalline, or hard rocks, like granite and basalt.  It remains an open question whether these endolithic microbes are metabolically active and therefore play a role in the regulation of the biosphere.  Our paper is a proof of concept, of how metabolic activity can be measured for microbes that live in subsurface hard rock environments.

Mark Nielsen working with the flow cells.

Photo of Mark working with the cylindrical flow cells that contained the indigenous microbes. The cells were connected to the aguifer via a borehole drilled in the tunnel wall. Natural ground water circulated from the aquifer, through the flow cells and back to the aquifer, all at ambient pressure. The flow cells were contained in refrigerated cabinets to maintain ambient temperature.

How were your experiments set up?

Initially, my idea was to measure the activity of the microbes through push-pull tests in the tunnel wall.  The idea was to inject a mixture of metabolic substrates and a tracer into the aquifer, let things incubate, and then extract back the fluids and look for consumption of the substrates.  But when I tried this it didn’t work – I couldn’t retrieve the compounds I’d injected because it turned out that there was a strong groundwater flow gradient moving away from the tunnel.  So instead, I moved my experiments to a flow system that had been installed in the tunnel a year previously.  The system allowed groundwater to flow out of the tunnel wall, into a sequence of flow cells where microbes could grow and samples could be taken, and then back into the aquifer.  I could then inject tracer and substrates into the system and monitor the growth and activity of the native microbes that had established themselves in the flow cells.  Because I could do counts of the number of organisms growing on the slides in the flow cells, I could calculate “cell specific rates” of metabolism – something that usually can’t be done in a natural setting. 

Where did the idea for the project come from and how did you get started?

I carried out the project during summer 2003 –  my first summer after I started grad school at OSU in the Subsurface Biosphere IGERT program.  One of the factors that had attracted me to the program was the availability of extra support for things like international travel.  I expressed interest in working internationally to my advisors, and Marty Fisk suggested working in the Äspö Tunnel and introduced me to Karsten Pedersen a scientist from Göteborg University, who directs the Deep Biosphere Lab there. 

When you look back, how was the project important to your development as a scientist and to your graduate school experience?

I had the support of my OSU professors, Marty Fisk and Jack Istok, plus my host scientist, Karsten Pedersen, but much of the time I worked independently.  So, for example, when my initial plan to run the experiments in the aquifer itself failed, I had to come up with an alternate plan of how to run the experiments in the flow cell system.  As a beginning graduate student, this level of independence was sometimes intimidating, but ultimately it was positive because it forced me to be creative.  When I returned to OSU, I worked with Marty to develop a proposal for related hard rock studies through the Ocean Drilling Program.  The whole experience really gave me a sense of the research process – from proposal development to experimental design and that will really benefit me in the future.

Before grad school I had worked as a geologist and a water rights engineer, so I didn’t really have a biology background.  This project and the IGERT program in general have really made me a believer in the statement, “This is a microbial world.”  Its one of the biggest lessons I’ve learned - microbes are important in so many aspects of life.