Subterranean ‘Microbial Dark Matter’ Reveals a Strange Dichotomy

The genes of microbes living as deep as 1.5 kilometers below the surface reveal a split between minimalist and maximalist lifestyles

Researcher wearing safety helmet and headlamp collecting a sample in an underground former gold mine

Magdalena Osburn, a geoscientist at Northwestern University, extracts microbe-laden water from a borehole in the Sanford Underground Research Facility, a science laboratory in the former Homestake gold mine in South Dakota. Such samples are revealing new details about the nature of life deep beneath Earth’s surface.

Devoid of light and deprived of nutrients, the depths of Earth might seem too barren to bother scouring for signs of life. But subterranean microbial organisms actually make up an enormous part of our planet’s biosphere. They are second only to plants in terms of total estimated biomass.

Now an abandoned gold mine in South Dakota is allowing the deepest look yet into this secret world of buried biodiversity. In new research published in the journal Environmental Microbiology, a genetic analysis of the mine’s microbes from as deep as 1.5 kilometers beneath the surface reveals a schism in survival strategies. Some microbes have big, bulky genomes that prep them to digest any nutrient that might come their way. Others are so genetically streamlined that they can’t even make some of life’s fundamental building blocks and instead rely on scavenging them or living symbiotically with other species.

“It was just cool to find that total dichotomy in survival strategy,” says Lily Momper, a consultant at the environmental and engineering firm Exponent and the paper’s first author. Similar results have been seen at the few other deep microbe observation sites around the world, Momper says. “We think this is probably a strategy in the deep subsurface in general,” she adds.


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The life that lurks deep in Earth’s subsurface may be an analogue for alien creatures eking out existence on other worlds; compared with our own clement orb, every other planet or moon in the solar system that could conceivably harbor life as we know it offers far less hospitable surface conditions. Yet below those harsh exteriors, despite the all-consuming darkness, an organism would be shielded from dangerous cosmic rays and warmed by geological heat. Such subsurface niches could be the default abodes for any life elsewhere in the solar system, if not the cosmos at large—which makes the hardy microbes hidden within our own planet of vital interest to astrobiologists. If anything now lives on Mars, for instance, odds are that it dwells belowground and looks and behaves much like the denizens of Earth’s depths.

The microbes underfoot also have importance closer to home. No one really knows the details of how carbon moves from the atmosphere and aquatic environments into the subsurface, says Karthik Anantharaman, a microbial ecologist at the University of Wisconsin–Madison, who was not a co-author of the new research but whose laboratory created a genomic profiling tool that was used in the study. “How do microbes influence that cycle? At what rate is carbon transferred?” Anantharaman says. Without those answers, a nuanced understanding of the carbon cycle and its enormous influence on Earth’s climate and habitability may be impossible.

The questions become particularly pressing, given that humans are hoping to mitigate climate change by injecting carbon dioxide back underground, a process called carbon sequestration. “A lot of those conversations are happening without an appreciation for the fact that microbes actually live underground and might be interested in messing with those processes,” says Magdalena Osburn, senior author of the new study and a geobiologist at Northwestern University.

The Deep Mine Microbial Observatory, a network of deep boreholes situated in what was once the Homestake gold mine in the Black Hills of South Dakota, is one of the few places on Earth where researchers can study these deep communities over long periods of time. “There are very few such deep boreholes,” Anantharaman says.

The mine, which closed in 2002, penetrates 2,438 meters deep. Since 2007 it’s been a multidisciplinary science lab called the Sanford Underground Research Facility, and it is now used primarily by physicists who are studying neutrinos and searching for dark matter particles. But there is another type of “dark matter” down there, Osburn says: microbes that have never been cultured in a lab. They are only known from their genetic detritus, snippets and parcels of DNA that researchers can sequence en masse from filtered groundwater and painstakingly reconstruct. Retrieving these precious samples requires descending deep into the mine in a wood-and-metal elevator cage.

“Your ears pop, and it gets really cold at first, but then it’s really hot when you get down there,” Momper says. “It’s in the 90-plus-degree-Fahrenheit [32-plus-degree-Celsius] range when you’re that deep. [The elevator] is really rickety and kind of terrifying the first time.”

Once at depth, the researchers tap into drilled boreholes to access fluid-filled fractures in the rock, filtering several liters of water from each to capture thousands upon thousands of individual microbial cells and their genes. In the new study, the team collected samples from depths of 244, 610, 1,250 and 1,478 meters (800, 2,000, 4,100 and 4,850 feet) and compared them with samples taken from a nearby creek on the surface.

The researchers then popped open the microbial cells and sequenced their genetic material together in one fell swoop. From this mélange, the team reconstructed the resulting genes into organismic genomes using software that detected overlaps between individual sequences. The approach was a bit like taking a shelf full of books, shredding them and then reconstructing them from the shreds, Momper says.

This method revealed genomes that were never seen before, indicating a plethora of new species hiding in the former gold mine’s depths. The researchers also found a large amount of diversity among the organisms. “The thing that popped out at us immediately is that they’re doing a lot,” Osburn says. “The metabolic capacity of these organisms is wide, so there’s huge potential for nitrogen and sulfur and metal cycling all over.”

Some of the organisms were minimalists, with genes for only a few very specific metabolic processes. These weren’t surprising to see in a nutrient-poor place such as the subsurface, Anantharaman says, because there is a metabolic burden associated with maintaining a big, energy-hungry genome. More surprising, he says, was the discovery of a second class of maximalist organisms. These organisms had the ability to metabolize chemicals that were not found in their environment.

This overpreparation is surprising because there is an energy cost to maintaining so many genes for so many metabolic abilities, Osburn says. But the “prepper” nature of these microbes may be an advantage in the subsurface. “Fractures open; fractures close; things mineralize,” she says. “Many of these organisms are just prepared for whatever energy source comes along.”

One advantage of the former Homestake mine is that researchers can return again and again to repeatedly sample the same boreholes. There are a handful of other long-term observational sites around the globe where scientists have sampled microbial dark matter, including in Canada, Sweden, Switzerland and Finland. It’s challenging to make valid comparisons between these sites, Anantharaman says, because they cover such a broad assortment of environmental conditions. That makes it hard to answer questions such as whether and how microbial diversity varies with depth.

One common pattern, though, is that most sites host a wide range of life. Osburn and her team are now looking at sequencing not just DNA but RNA, the molecular go-between for genes and proteins. Studying microbial RNA can reveal not just what microbes can do, Osburn says, but what they are doing at a given moment. Another current project is analyzing subsurface biofilms—stable accumulations of microbes that are protected by slimy excretions, which we more typically encounter as scummy deposits in toilets and kitchen sinks. Biofilms are hard to study, Osburn says, but the researchers got lucky: They set up a long-term filtration system in a mine borehole in December 2019 and planned to collect it three to six months later. Instead COVID hit, and the filtration system sat for four years before the team could get back to check on it. Miraculously, it was intact.

“This is our closest approximation yet of what that in situ biofilm-based biosphere looks like,” Osburn says. “[The organisms] generated a lot of biomass, and it looks really different in a way that I’m excited about.”