Thousands of feet below the Earth’s surface seems an unlikely cradle for life. Indeed, for a long time, scientists wondered if any life could survive in such a hostile, pitch black, oxygen-poor environment.

Working in the depths of a gold mine, Maggie Lau (Left) and Rachel L. Harris (Right) filter water to capture microbial cells onto a filter membrane. Image courtesy of Rachel L. Harris (Princeton University, Princeton).

But about two decades ago, researchers found the first substantive evidence of organisms living deep underground. A Department of Energy drilling project had recovered rocks from more than a mile below the Earth’s surface in the Taylorsville Basin in Virginia, and scientists were surprised to find that these rocks harbored bacteria. More recently, researchers went looking for microscopic worms in deep mines, and discovered a variety of eukaryotic organisms living at these depths (1, 2). Most of these eukaryotes survive by eating deep-living bacteria.

But there’s still a lot we don’t know about how the bacteria themselves survive in an environment without sunlight or plentiful oxygen for energy. Recent work, based on samples painstakingly retrieved from deep gold mines, appears to provide a crucial clue: the bacteria work together in intricate networks, cooperating to survive.

“We have long understood that life exists in the deep subsurface, but we don’t actually know what they’re doing in that environment,” says Maggie Lau, a geomicrobiologist at Princeton University and the study’s first author. Lau and her colleagues identified and analyzed the metabolic networks used by deep-living bacterial communities. By capturing the “metabolic landscape” of these underground environments, the authors showed that the bacteria use a wider range of energy sources than expected, and work together to survive (3).

Studying these kinds of metabolic landscapes will be crucial for understanding deep-living bacteria, says Princeton University geomicrobiologist Tullis Onstott, the study’s senior author. “It’s basically getting an image of what is actually active down there,” he says. “It’s like catching them in the act.”

Low-Energy Living To examine deep-living bacterial communities, researchers either use drills to collect samples or travel to mines and caves that provide access to these depths. Onstott studied deep life in several gold and diamond mines in South Africa. To collect samples for the current project, Lau ventured nearly a mile underground in South Africa’s Beatrix gold mine. Getting to the study site is an odyssey in itself. Lau takes a mine cage down about a mile, and then walks another half-mile or more, sometimes wading through flooded tunnels, all while carrying a heavy backpack. She often relies on a headlamp to illuminate her work area in the pitch-black depths. To collect bacterial samples, Lau filters water from boreholes—tubes drilled into the ground to look for oil or minerals, which sometimes fill with underground water—at her subterranean study site. Because this part of the Beatrix gold mine has no active mining, the borehole served as a fount of uncontaminated samples. To collect enough samples for her analysis, Lau had to filter nearly 23,000 gallons of water over 15 days. “Getting good samples is always the biggest challenge,” says Onstott. “We really need to sample large volumes because the biomass concentration is so low.” Only a small fraction of deep-living bacteria have been grown in laboratory cultures, limiting what researchers can learn about these organisms through traditional microbiology approaches. Advances in DNA sequencing technologies have helped offer insights into the bacteria’s metabolic pathways. But such metagenomics studies aren’t sufficient to find out what these organisms actually do in the depths. Researchers need to examine the bacteria’s RNA and proteins to determine when and at what levels their metabolic genes and proteins are expressed. “When it comes down to constructing a model for how the organisms that are present may or may not be active, or how active they are, and what they contribute to the system, you really need to go beyond metagenomics and get a proteomics and transcriptomics approach to address that,” says Oregon State University geomicrobiologist Frederick Colwell, who was not involved in the study. The first step is to gather enough RNA and protein from deep-living microbes, a challenging feat in these low-biomass systems. Lau took advantage of her study site to collect large amounts of uncontaminated samples and devised methods of preserving the samples for her analysis (keeping them cold at all times, for example). As a result, Lau was able to gather enough high-quality DNA, RNA, and proteins for metagenomics, metatranscriptomics, and metaproteomics assays, offering a revealing look at the metabolic genes and proteins expressed in these communities. Lau and Onstott found that different groups of deep-living bacteria paired up, with each eating substances produced by the other and producing the “Cooperative functioning in the subsurface seems to have benefits to enable the system to be more stable and more diverse than we thought.” —Maggie Lau other’s food in return. This type of cross-feeding is called “syntrophy,” and may help bacteria survive these harsh environments. “In the subsurface environment, not many energy-producing processes are favorable,” says Lau. “In order to live here, organisms work with each other to make conditions favorable,” she says. “The organisms that they are detecting are not new to science, but their collusion in this sort of system, to me that’s pretty novel,” says Colwell. Among the metabolic pathways expressed in the deep subsurface are those involving nitrogen (Upper Left), sulfur (Upper Right), and carbon (Below). Black and gray solid lines indicate reactions mediated by enzymes; black dotted lines in the nitrogen cycle indicate abiotic reactions. Reproduced from ref. 3.

Microbial Menu When Lau and Onstott analyzed their data, they were in for another surprise. The researchers had expected hydrogen, methane, and sulfate to be the major energy sources. “Because of the abiotic processes that can generate these energy sources, people think reactions that utilize these resources should be prevalent,” Lau explains. Instead, only a minority of the deep-living bacteria fed on these chemicals. This minority produced sulfur and nitrates as metabolic byproducts, which then fueled the vast majority of the bacteria that live at these depths. Previous geochemical work by Onstott suggested that denitrification—the reduction of nitrate by microbes—might be occurring in these underground communities (4). “Lo and behold, our data shows that nitrate is a very important source of energy in the deep subsurface,” says Lau. Nitrate is usually found at pretty low concentrations in these environments, and Lau suggests that bacteria may be keeping nitrate levels low by feeding on it. Bacteria feeding on different substrates appeared to work in syntrophic pairs. For example, bacteria that eat sulfate produce sulfide. This sulfide becomes the food source for other bacteria, which produce sulfate and complete the cycle. Another bacterial pair may oxidize and reduce methane. By pairing up, bacteria can make use of a greater range of energy sources than would otherwise be feasible in these barren environments. “Cooperative functioning in the subsurface seems to have benefits to enable the system to be more stable and more diverse than we thought,” says Lau. “It’s really a good way to think about it, as a bunch of syntrophies, rather than competing reactions,” says Karen Lloyd a geomicrobiologist at the University of Tennessee who was not involved in the study. The study’s findings could expand the kinds of processes and organisms researchers look for when looking for life. “Their work is relevant to people working in very different environments,” says Lloyd.