A decade of extremophiles

When the team from the Deep Mine Microbial Observatory, or DeMMO, left in December 2019 after a routine visit to collect samples and install incubation experiments at the Sanford Underground Research Facility (SURF), Dr. Magdalena Osburn expected her team would return to South Dakota to gather experiments the following spring.

“We were going to come back in six months,” Osburn explained. “We want them [the microbes] to colonize our experiments, and they’ve had four years to do it. You literally cannot plan that experiment.” Osburn, the experiment principal investigator for DeMMO and associate professor at Northwestern University, studies subsurface geochemistry and microbiology, and last visited SURF in 2016.

Three researchers from Northwestern University, including Osburn, finally returned to SURF last week, marking ten years since Osburn and the DeMMO scientists began studying extremophiles at SURF. Now they were coming to check on the status of their experiments, and, optimistically, to start again from where they left off.

“In addition to picking up the stuff that was left, we’re essentially beginning anew at DeMMO and setting our baseline geochemical expectations, microbiological expectations, and using that to shape where we go from here," said Osburn.

“Assuming that things look pretty similar, now we are in a place that we can delve into more specific questions. We’ve got the framework but now we can really start to develop those more specific hypothesis, getting into more specific process. We know who’s there, but why? What is it that they’re doing? What makes those organisms so specially adapted to the subsurface?”

Osburn prepared to take samples and assess conditions, but also observe if the changes within the underground, including the excavations for the Deep Underground Neutrino Experiment (DUNE) have impacted their sites. Osburn explained the sites were chosen to sample the outer hydrological footprint. “We will make a suite of measurements on this trip that will tell us an answer if things look the same. We will know an answer for each site definitively. We’ll have the before and after data to say, ‘Alright, how you doing DeMMO 4? Are you the same site you were before or not?’”

When Osburn and researchers from Northwestern, as well as researchers from other institutions, began setting up DeMMO in 2013, the initial plans were to determine the suitability of SURF for long-term studies. In 2019, Osburn published research describing SURF as a geochemically stable portal into the deep subsurface. That stability not only provided research sites for obtaining samples, but an environment to wait patiently for her post-pandemic return. On their first day, Osburn visited sites on the 2000-foot level and the 4100-foot level.

“On the 4100, that flow is down – it is a down hole, so it flows up out of the hole – it’s flowing less than half what it was before. That was a little surprise. So, that site was looking a little bit different, but everyone else is pretty much as I remember just from my first pass looking at the chemistry. It’s within what we measured before. The water coming out of those fractures is the water coming out of those fractures. It’s doing its thing.”

The plan upon return was for cleaning and straightening up and collecting what biomasses grew in the subsurface. “How pristine it is remains to be observed,” said Osburn ahead of her first descent.

Accompanying her on the DeMMO research last week were Dr. Bradley Stevenson, research associate professor at Northwestern University and third-year graduate student Meera Shah. The three set up their above ground location optimistic about obtaining both solid and liquid samples to take back to the Chicago area for analysis. At the end of their three days, their collection of samples, plus the gear they stored at SURF during their absence, filled the room completely.

“We’re collecting a lot of samples, so the very next steps are starting the processing pipeline for some of the things we are going to analyze right away, some of these things we’re going to keep and look at the results of those first analyses and make a plan.” The tubes, jugs, and vials received labels identifying their dates, collection points throughout SURF, and important information to recall over the months of research awaiting them.

Shah anticipated the long-term project back at the university lab. “After this trip, there’s plenty of samples to work with so a lot of lab work and analyzing. I do enjoy that work, so I’m looking forward to if we find any trends or patterns, or what these samples tell us.”

Shah’s focus will also be on the amino acids in both the biomass solids and the liquids harvested from the underground sites. “I think the biggest question for Meera is in addition to these solid samples that have biomass on them, what we spent most of our time underground doing is filtering water to collect cells for proteins and lipids, and DNA,” Osburn explained.

“The question is, we’ve never tried to do proteins on these filtrates before and we don’t really know how many cells are in the water currently, so did we get enough? If we did not get enough, then we’re coming back. By the spring, we’re likely to know how well we did and what strategies we need to implement,” said Osburn and Shah’s studies are expanding the knowledge gleaned from the existing sites.

Shah’s studies aren’t the only research that the next decade will be generating on this newest round of sampling and collecting. Stevenson also bring a fresh perspective to the science. “He had a skillset that was really complementary to leverage. He’s a real microbiologist where I’m trained as a geologist and a geochemist. Essentially our strengths are coming from the opposite sides of geobiology and so it makes a more complete working picture and strength in more areas. I refer to us as a supergroup.

In addition to supporting the existing research, the team obtained samples for future studies of actinobacteria. “They grow on the rock walls. Quite often when you scrape them off, the rock is really degraded, crumbly. They are also found in caves and they’re distribution is interesting. We think it has to do with air exchange – where fresh air comes in perhaps,” explained Stevenson.

“I have studied them in the past in lava tubes and limestone caves,” Osburn confirmed. Despite missing out on collecting samples during the past four years, she discovered that the break allowed her to turn her research in new directions, including studies beyond DeMMO, like actinobacteria. “It’s sort of a cave feature and I don’t know why I wasn’t clued into them in the mine, but part of having a little break in DeMMO is I’ve been doing cave work so when your mind learns to see things, you see different things.”

Osburn explained that during her years away from SURF, the subsurface systems that exist within the Black Hills prompted her to think more broadly when seeking out other locations for field work. “Because I couldn’t come here, I went to Mammoth Caves and started this large-scale microbiology of caves, learned to identify all the actinomycete biofilms and some of the interesting features that you get in caves – things like sulfur seeps, things like iron seeps. It turns out it’s not that different than the shallower levels of SURF. So, I’m thinking on a larger spatial scale of the depth gradients and the space gradients. Putting this larger scale vision together has been the culmination of the ten years.”

The decade of successful research for DeMMO continues to be measured in a variety of ways. “I think it’s a miracle that we’re back here sampling the same sites ten years later. That’s success to begin with. I think we’ve learned a phenomenal amount about subsurface geomicrobiology from looking at these systems, and being able to come back to the same systems again and again is really unique in subsurface geomicrobiology.”

Likewise, Osburn gave credit to the subsurface biomes still to be explored at SURF. “Most other places you don’t have the liberty of coming back time after time.” She sees her break in DeMMO research as an unexpected benefit towards her overall success. “It’s a unique perspective that we can gain. The concept that we’ve had things incubating and growing for literally years, is unheard of.”