In underground caves and equally extreme environments, Jenn Macalady finds analogs for life on other Earths.

The first time she entered Italy’s Frasassi Caves, Jenn Macalady gave her husband a scare. “He was waiting for me to come out,” she remembers. “I was so amazed by what was down there that I lost all track of time. I was completely enthralled.”

formations inside of cave
Brian Kakuk (Bahamas Underground)

A microbial biofilm creates an ancient and otherworldly landscape deep within Magical Blue Hole, a flooded cave on Abaco Island, The Bahamas. Light harvesting bacteria in the biofilm thrive despite the dim light and absence of oxygen in the cave water.

Frasassi, discovered in 1948, is one of the show caves of Europe. The 19-mile subterranean system comprises one of the largest and most spectacular limestone complexes in the world, drawing thousands of visitors every year. But it wasn’t the stalactites and stalagmites that captured her. Macalady, a geomicrobiologist at Penn State, was more interested in the slime.

Biofilm would be the polite term. Biofilms are complex layered communities of microorganisms that form on all sorts of surfaces, literally everywhere there’s enough moisture: in bathroom showers and on stagnant ponds, on boat hulls, even on the surface of your teeth. Biofilms are particularly adept at colonizing extreme environments, from hot springs to glaciers.

In Frasassi, they exist on damp walls and in the pools and streams of an underground aquifer, entirely without light and mostly without oxygen. Yet these self-contained ecosystems have found a way not just to survive, but to thrive. In fact, their presence plays a vital role in the cave system’s continuing formation. And for Macalady, the underground environment they inhabit is a rare working model of early Earth.

When Microbes Ruled

For the first two billion years of Earth’s history, the planet’s atmosphere was completely devoid of oxygen. Without this crucial source of energy, there was no opportunity for the development of multicellular life. “Microbes ruled the world,” Macalady says.

edge of stream in cave
Jenn Macalady

White microbial biofilms create intricate patterns in response to stream flow in the Frasassi cave system, Italy. DNA sequences from this biofilm and dozens of others are being analyzed to discover how microorganisms eat and breathe sulfur chemicals.

Oxygen first made its appearance as a waste product, when prokaryotes—simple, single-celled organisms—evolved the capacity for photosynthesis. Initially, it was quickly dissipated by rock weathering, chiefly the oxidation of iron, but gradually the stuff began to accumulate in the atmosphere. Then suddenly, about 2.5 billion years ago, in what scientists call the Great Oxygenation Event, atmospheric oxygen rose dramatically to about ten percent of the present level.

“This would have resulted in a surface Earth that was largely micro-oxic, like the Frasassi aquifer,” Macalady says. “So chemically this cave environment is very much like what could have been on the surface of the Earth for a billion years.”

Specifically, Frasassi represents what she calls a “sulfur world,” an ecosystem based not on oxygen, but on sulfur as an energy source. As Macalady explains it, sulfur is present in abundance because of a thick layer of gypsum at the bottom of the cave. Water coming in contact with the gypsum dissolves it to form hydrogen sulfide. The sulfide mixes with small amounts of oxygen to form sulfate, and “about half” of the microbes in the biofilms use this form of sulfur for energy. “They’re basically breathing sulfate instead of breathing oxygen,” she says. In turn, these microbes produce sulfuric acid as waste, which eats away at the limestone walls, deepening the cave by a couple of inches every thousand years.

gormations inside of cave
Jenn Macalady

White microbial biofilms coat the bed of a stream hundred of meters below the ground surface in the Frasassi cave system, Italy.

Understanding the workings of this system, Macalady says, offers clues to how early life evolved. And, in addition to being windows onto Earth’s history, she says, these environments “have turned out to be really extraordinary model ecosystems for microbial ecology in general.” Within the layers that make up a biofilm there are many different types of microbe species, each playing a different role in the community, she explains. Through genetic analyses, “we are trying to map which organisms live where in the caves and how they correspond to the geochemical environment.”

Two years ago, Sharmishtha Dattagupta, then working as a postdoc in Macalady’s lab, discovered a symbiotic relationship between a sulfur-breathing microbe and a tiny cave invertebrate living in Frasassi, the first time such a relationship has been seen outside a deep-sea hydrothermal vent.

In fact, “You could think of these caves as hydrothermal vents on land,” Macalady says. Like the sea-floor vents, they rely on chemical energy to support life. And like hydrothermal vents, they are geochemical “islands” that allow for tests of gene flow between isolated microbial populations.

The Right Tools

woman and man in orange jumpsuits and helmets working inside cave
Lubos Polerecky (Max Planck Institute for Marine Microbiology)

Jenn Macalady (left) and Ph.D. student Daniel Jones collect deep groundwater from the Frasassi cave system, Italy to learn about chemicals supporting life in the absence of light and oxygen.

Parsing the details of these exotic communities depends entirely on the still relatively new approach known as metagenomics, by which the hash of genetic material taken from an environmental sample can be painstakingly untangled in the lab and reassembled into its constituent genomes.

“This ability is allowing us to learn by leaps and bounds about the microbial world,” Macalady says. But the ever-accelerating advance of the technology brings its own challenges, she notes. “Sequencing is now so cheap that we’re just drowning in data. In a soil ecosystem, there are tens of thousands of species per gram. It’s really intractable.”

Because biofilms are isolated communities, however, they are relatively simple. Relatively being the operative word. “We’re still talking about thousands of species,” Macalady grins. “Just not tens of thousands. But simple is better for us because we like to be able to dissect the system well enough to make the links between the environment and the microbes,” she explains. “It’s exciting to be able to link a microbial species with a chemical reaction that’s occurring in the environment. If we can make these links we can start to understand the behavior of the system as a whole.”

She and her team received an important boost when the Department of Energy’s Joint Genome Institute agreed to fund a recent proposal. “They are offering to sequence for free something like 40 or 50 samples from the Frasassi aquifer,” Macalady explains. “It amounts to gigabases of data—You’d need an army of postdocs to make sense of it.”

Into the Blue

In the fall of 2008, Macalady got a phone call from Kenneth Broad, a cultural anthropologist at the University of Miami who also happens to be one of the world’s foremost cave divers. Broad was leading a National Geographic Society expedition to the Bahamas, to explore the underwater caves known as blue holes. He needed a microbiologist. Macalady smiles at the memory. “I think he saw from my Web site that I was ‘expeditionary,’ as they would say. So he called me.”

woman inside cave holds up a piece of paper
Clara Chan (University of Delaware)

Macalady displays a "photograph" created by microbial activity in an oxygen-free cave pool in the Frasassi cave system, Italy. The black deposits on the film reveal the location of sulfur-breathing bacteria within the pool sediments.

Inland blue holes are spectacular submarine environments, flooded limestone caves which, because of their isolation from tidal flows, have unusually stratified water chemistry. In a typical blue hole, a freshwater cap provided by rainwater sits atop a denser layer of salt water that may extend down 500 feet or more. The freshwater acts as a barrier to atmospheric oxygen, preventing the decay of organic matter in the salty depths. The anoxic murk, in short, is a treasure trove of fossils.

Because of their inaccessibility, however, these sites remain largely unexplored. The idea behind the so-called Bahamas Blue Hole Expedition was to gather a large team of scientists and experts who could tackle this terra incognita. Their exploits over six weeks in the summer of 2009 resulted in a cover story in National Geographic and a PBS NOVA documentary.

The lion’s share of the coverage was given over to the extreme danger of cave diving and the potential for fossil rewards. Macalady’s focus, though, was on what remains alive in that harsh environment, specifically the bacteria that survive there without oxygen. She was particularly interested in the creatures that live in the anoxic layer, where conditions persist that are exactly like those thought present on Earth some four billion years ago. “The question is, without oxygen, how do these microorganisms make a living?” she says.

“There’s a surprising amount of overlap between the anoxic seawater that’s in blue holes and the anoxic layer of the Frasassi aquifer. The distinguishing feature is that blue holes are cave systems with skylights. The sinkhole is the skylight where sunlight enters the system. That allows a different group of organisms to survive.”

In the Bahamas Macalady collected samples at many depths, and from these she has already recovered DNA from hundreds of previously unknown species of the type that dominated early Earth. “To give an idea of just how unique each hole is,” she told National Geographic, “we analyzed the DNA of microbes from five inland blue holes and didn’t find any shared species.”

That haul has left her with “years of analysis” left to do, Macalady admits. Yet she and Broad are already “talking about our next scheme,” involving exploring a cave system in the Dominican Republic. “There’s a little bit more contact with the atmosphere in these caves, enough oxygenated water entering that you get a nitrogen world instead of a sulfur world,” she explains. “As a comparison this is too good to pass up.”

Out of this World

Last fall, Macalady and Lee Kump, a geochemist at Penn State, led a field workshop to fathom yet another type of low-oxygen environment. The site was Fayetteville Green Lake, near Syracuse, New York.

Because of its depth and the presence of salt deposits at its lower layers, Macalady explains, Green Lake “is stratified in the same way that the blue holes are stratified.” Its warmer, fresher shallow layers don’t mix with the colder saltier layers below.

woman in sleeveless shirt holding equipment and smiling
Jill Heinerth

Jenn Macalady prepares to collect a microbial sample from anoxic water in Sawmill Sink (Abaco Island, The Bahamas) during the 2009 National Geographic Blue Holes Expedition.

As a result, the top 20 meters or so are oxygenated, and the layers below are anoxic, home to the same types of sulfur-breathing bacteria that populate sulfidic caves. “One of the things going on is organic matter falling into the lake falls to the bottom and is decomposed. And that decomposition uses first all the oxygen and then all the nitrogen and then it begins to use the sulfate. So the oxygen breathers use up the oxygen, the nitrate breathers use up the nitrogen and then the sulfate breathers kick in, all chewing on this decomposing organic matter.”

In turn, she says, the sulfate breathers make hydrogen sulfide as a waste product, which then fuels more photosynthesis in the sunlit layer. “So there’s a complete sulfur cycle in this lake.”

This unusual geochemistry makes Green Lake a good place to study the early days of photosynthesis. “This lake in particular has been proposed as an analog for the second half of the Precambrian,” Macalady says. Indeed, Kump has used its example to suggest a reason for the great Permian extinction, which killed 95 percent of all species on Earth. In Kump’s view, the Permian Ocean may have been very much like Green Lake. The gradual build-up of hydrogen sulfide in the lower levels could have become so intense, he reasons, that the poisonous gas eventually escaped into the atmosphere, wiping out animals and plants alike.

Green Lake, like the Bahamian blue holes and the Frasassi aquifer, is not just a model of early Earth, Macalady stresses. Conditions in these areas may also mimic present-day life on other planets, yet-to-be-discovered environments where water is present but oxygen is lacking. Indeed, her work in all three areas has substantial funding from NASA’s Astrobiology Institute. As she told National Geographic, “The universe is made of the same elements, and habitable planets are likely to share many of the same characteristics, like a temperature range conducive to life and the presence of water.”

The topic brings a fresh gleam to her eye. “I find that astrobiology is something that I love thinking about, I love talking to other people about,” Macalady says. “It’s by nature interdisciplinary, and really it is sort of mind-exploding. You’re returning to basic physics, basic chemistry, fundamentals of biology, because you’re thinking about other worlds where the rules might be different, but at the same time there are some rules that we know can’t change.

“You end up going back to the roots of science, recombining all the rules in different ways, and thinking about ‘If this type of world were to exist, how would you know? How would you go about finding the presence of life?’ ”

Jennifer Macalady, Ph.D., is associate professor of geosciences in the College of Earth and Mineral Sciences; jlm80@psu.edu.

Last Updated October 26, 2011