It’s December 2018. Karthik Anantharaman awakens at 6 a.m., afloat in the middle of the Pacific Ocean. He’s barely slept, adrenaline is flowing. There’s little time, and he and Alvin need to get ready.
Anantharaman goes through a series of checks — a blood pressure measurement, a health status questionnaire — to make sure he’s fit for what’s ahead. He eats breakfast at precisely 7:15 while Alvin gets connected to several harnesses. Weather patterns are monitored. At 8:00, he and Alvin finally meet up. They dive from the deck of the R/V Atlantis and slip beneath the waves.
Alvin is a small submarine. Its job is to carry Anantharaman, along with another scientist and a pilot, down to the ocean floor.
The first few hundred meters of the slow descent are uneventful. Waves and currents rock the small vessel, but light still penetrates the water here. The craft has no downward propulsion, so weights are used to pull it into the depths. Around 300 meters down, darkness starts to close in, and the pilot turns off Alvin’s lights. The passengers are immersed in black.
That’s when the light show starts.
All around them, thousands of bioluminescent organisms appear as small, gleaming figures in the dark water. “It’s unreal,” says Anantharaman, an assistant professor of bacteriology. “It’s just one shape after another glowing an incredibly brilliant blue. It’s all the way through the water column, and it’s one of the most brilliant sights you can imagine.”
But the dazzling display is just a perk of the trip, not the destination. They’re heading toward the East Pacific Rise hydrothermal system, where they will navigate the ocean floor and collect samples near deep-sea fissures that discharge water heated by the earth’s interior. The samples will be sent all the way back to Anantharaman’s lab on the UW campus, where he and his team will try to unravel the mysteries of sulfur metabolism as it’s carried out by the bacteria and viruses that live deep in the sea.
Anantharaman calls himself and the members of his lab “microbial ecologists.” They use DNA sequencing, data analysis, and even in-house bioinformatic tools — they developed their own software called VIBRANT and METABOLIC — to study bacteria and other microbes in both environmental systems and humans. Their goal is to better understand sulfur metabolism in multiple systems.
Metabolism is the formation and use of energy required for life. In humans, this involves compounds such as carbohydrates and oxygen. But bacteria are more versatile. They can use other elements, such as sulfur. In the absence of light at deep-sea vents, higher organisms rely on sulfur-transforming bacteria. Tube worms, clams, mussels, and other animals that have lost the ability to eat host these symbiotic bacteria, relying on them to consume sulfur and produce the energy they can’t make themselves.
This arrangement can be found in ecosystems that thrive near deep-sea hydrothermal vents.
But the origins of Anantharaman’s adventure into the depths lie nowhere near the ocean. It began when he was studying iron compounds and nanoparticles during a civil and environmental engineering master’s program at the University of Michigan.
“We use engineered iron nanoparticles to treat pollutants, but when you think about it, it’s microbes that produce [the nanoparticles] in the environment,” explains Anantharaman. “I had this awakening moment where I thought, why am I thinking about synthesis and chemistry? I want to study the biology of how these bacteria are actually producing these nanoparticles in nature.”
With that realization, Anantharaman decided to pursue a Ph.D. in microbiology. But as he started down his new academic path, the field began to change. DNA sequencing exploded. It was being used extensively to study microorganisms such as bacteria. The computational skills Anantharaman acquired during his engineering training became very useful when he started using DNA sequencing to find microorganisms that metabolized sulfur. That’s what led him to hydrothermal vents on the bottom of the ocean.
Hydrothermal vents are an ideal place to study microorganisms and ocean biology. The vents release trace elements, such as iron, manganese, zinc, and copper, which are often carried by currents for hundreds or thousands of miles. These elements are extremely limited in the ocean environment, and they are necessary in the surface ocean for photosynthesis by bacteria and algae. These algae, in turn, are the base of the food chain for nearly all ocean life. Hydrothermal vents, therefore, control ocean chemistry.
The vents are also perfect places to study sulfur metabolism. They serve as conduits for energy and chemicals that travel from deep within the earth into the oceans. Consequently, hydrothermal vents have some of the highest levels of naturally occurring sulfur compounds on the planet. Over billions of years, these high concentrations of sulfur have resulted in the evolution of microorganisms that can utilize the compound for generating energy.
“This is a unique ecosystem that does not depend on the energy source of light, unlike most other ecosystems on the planet,” says Zhichao Zhou, a postdoctoral fellow in Anantharaman’s lab. “It is a chemosynthetic ecosystem that is solely dependent on the chemicals brought out from the vents.”
Although it’s a useful energy source, sulfur can also cause a lot of problems in biological systems. The sulfur compound that’s produced by hydrothermal vents is hydrogen sulfide, a toxic gas that smells like rotten eggs. It’s poisonous to aquatic organisms and humans alike. In the environment, microorganisms can metabolize hydrogen sulfide into non-toxic compounds such as sulfate, but it’s an intricate process that science hasn’t fully apprehended.
“A lot of microbes can use sulfur compounds, and it’s a cycle we really need to understand,” says Anantharaman. “If we want to study sulfur metabolism and look at the diversity of organisms associated with this process, the hydrothermal vents are a great environment to do that.”
Anantharaman has taken two separate trips to hydrothermal systems. In addition to East Pacific Rise via the R/V Atlantis, which is owned by the U.S. Navy and operated by the Woods Hole Oceanographic Institution, he also journeyed to the Guaymas Basin aboard the Schmidt Ocean Institute’s R/V Falkor in March 2019. Onboard, he collected samples with the assistance of a submersible robot named ROV SuBastian rather than a manned submarine. Both vessels sail from Manzanillo, Mexico.
As locations for study, the vent systems are ideal, but the research process is not. Collecting samples from hundreds of meters under the ocean is incredibly difficult, and processing everything collected at those sites is another enormous undertaking.
Back inside Alvin’s close quarters, Anantharaman and his colleagues first gather animals such as snails, worms, clams, and mussels. Then the researchers collect mats of bacteria that are oxidizing sulfur compounds. And finally, they filter fluids.
Since this step takes the longest, it’s left for last. Due to oxygen limitations, passengers can stay in Alvin’s confines for only eight hours at a time; and even in that brief stay, carbon dioxide concentration can get up to 1% in the submarine (compared to atmospheric levels around 0.03%). So any time remaining on the bottom of the ocean is spent filtering fluid, looking for the bacteria and viruses in the water that are oxidizing sulfur. The dive day is long and intense. Researchers go directly from collecting one sample to the next while following a carefully designed plan.
“I had taken a sandwich and a couple of energy bars and water,” Anantharaman says. “You don’t get a break; there is no lunch break on the submarine. You just keep sampling and eat on the go. Usually one scientist is taking notes while the other has eyes outside.”
Once sampling is complete, it’s time to start shutting things down. Alvin and the researchers are to be on the surface by 5 p.m., and the ascent takes a bit more time and control than the descent. Before heading to the surface, several safety checks must be conducted, and communication has to be established with the ship. A slow rise is important: The submarine contains sensitive scientific samples stored on the outside that could become loose or damaged.
During this particular dive, Anantharaman and his colleagues have a fun opportunity before they reach the surface. They speak, via satellite, to attendees at the American Geophysical Union conference about their work. Undergraduates and graduate students ask questions as they watch the dive team, with hydrothermal vents in the background. “It was one of the most memorable sites I can imagine, and that was a fantastic conversation,” Anantharaman recalls.
Finally, at 3:30 p.m., the Alvin crew drops the weights that hold the sub on the ocean floor, and it begins to rise. After about an hour, they reach the surface. Typically, a boat attaches to the submarine, stabilizes it, and leads it back to the ship. But after the short delay from talking with students at the conference, the boat has drained its battery. Anantharaman and his colleagues are stuck inside Alvin.
“We had to wait, and the surface was very choppy,” says Anantharaman. “We survived, thanks to Dramamine, but we were not in good shape when we got out of the submarine. We had waves really bashing us for about an hour.”
With that ordeal behind them, there is still no time to rest. Once back onboard the R/V Atlantis, which is one of the most sophisticated research vessels afloat, all the samples need to be processed and stored. Finally, autonomous vehicles take over during the night and map the sea floor. Bottles are also dropped over the side of the ship during sleeping hours to collect water for further filtration and measurements, such as oxygen and pH levels and temperature.
Even when using a robot, such as SuBastian, rather than sending scientists down in a submarine, the research is still exceptionally time-consuming. The robot begins its dive at 6 a.m., and scientists are awake well before that completing preparations. Once the submersible starts sampling, it’s all hands on deck as researchers keep a constant eye on cameras and computer screens. While the manned submarine can only dive for about eight hours for the safety of the passengers, the robot is not constrained in any way. SuBastian can stay underwater for 24 hours or more.
“Fieldwork in the ocean is quite challenging,” Anantharaman says. “What exacerbates that on a ship is that there’s a limited number of scientists. Everyone’s trying to do their own research, and you don’t get too much help. You just have to suck it up and work your 16- or 18-hour days.”
At the lab in Madison, Anantharaman (after a much-deserved rest) works with his team to further process the microorganisms and viruses he filtered from water at the hydrothermal vents. They extract DNA and RNA and sequence them. They apply bioinformatics — the use of computational technology to analyze large sets of biological data — to reconstruct the genomes of nearly every bacterium or virus in the sample. From there, they can start to decipher what sort of metabolism those organisms might be carrying out in the deep oceans.
“I am working to characterize the microbial community based on the genomes of microbes and to investigate how these microbes transform energy and elements in the hydrothermal plume,” explains Zhou. “Based on the data, I can investigate the function and activity of these microbes at a very high resolution.”
The team also works to analyze the data collected from a custom filtration system that Anantharaman takes on the ships. The system allows them to load three different filter sizes. The largest filter collects algae and large bacteria, the middle filter catches most of the remaining bacteria, and the small filter snags viruses. This on-the-spot filtration is necessary because bacteria can change their activity in very little time, making it vital to process samples promptly.
“We used the filtration system, which we named HIFIV [pronounced “high five”], on both of the cruises, and it worked fantastically,” says Anantharaman. “We are now in the process of analyzing a lot of data, and we’re very excited to see what results we find.”
Anantharaman and his lab are discovering new bacteria and, along with them, the processes associated with sulfur metabolism. They hope that this knowledge will transform scientists’ understanding in a variety of areas, including low oxygen environments, which can be important contributors to climate change; the role of sulfur metabolism in human health; and, through discoveries of new microorganisms, the intricacies of the tree of life.
Of course, sulfur can be found in many locales beyond deep-sea hydrothermal events — anywhere life exists, really. So Anantharaman and his team are studying sulfur cycling in other environments, including freshwater lakes and even the human gut.
Anantharaman’s interest in the digestive tract stems from his postdoc position in the lab of Jillian Banfield at the University of California, Berkeley. There, he saw how concepts used in environmental microbiology can be applied to human health.
Specifically, Anantharaman is interested in gastrointestinal disorders such as colorectal cancer and Crohn’s disease. If hydrogen sulfide is formed in the gut, it can throw things out of sync and exacerbate both conditions. Using the deluge of publicly available data related to the human gut, his team has identified a number of sulfur pathways that could be contributing to human disease.
They are now expanding these studies through a collaboration with a group of University of Illinois scientists who study colorectal cancer. Using their cohort of patients, Anantharaman’s lab is uncovering how sulfur compounds are being produced and how they affect the progression of the cancers. They are also finding interesting viruses that may be associated with producing hydrogen sulfide or killing beneficial bacteria and therefore exacerbating cancer.
“I utilize data generated from DNA sequencing to determine what these viruses are doing, which microbes they infect, and how this can affect the health of the planet and impact humans,” says Kristopher Kieft, a microbiology graduate student in Anantharaman’s lab. “I’m primarily interested in how viruses can manipulate how bacteria metabolize sulfur. When possible, I also cultivate viruses in the lab to measure any effects on sulfur metabolism.”
Another graduate student, Patricia Tran, is taking on the lab’s efforts on the freshwater front. A Ph.D. candidate in freshwater and marine sciences, Tran is looking at both Lake Tanganyika in Africa and Lake Mendota, right on campus. The latter is often called the most studied lake in the world.
“[Bacteriology professor] Trina McMahon and her group have been studying the lakes for years. There is a lot of microbiology and chemistry data,” says Anantharaman. “But what was missing with Lake Mendota, and where we saw an opportunity, was an understanding of sulfur and viruses in the lake.”
Phosphorus is widely analyzed in lakes because of its role in eutrophication — the presence of excess nutrients in water bodies due to runoff — and the problematic algal blooms that stem from it. Sulfur plays a role in controlling phosphorus, and Anantharaman and Tran want to understand that process better. They are sequencing the genomes of both the bacteria and viruses from samples of Lake Mendota, and they aim to tease out how those microorganisms might be associated with sulfur cycling. To filter those samples, they’re using the same HIFIV system that was used in the ocean.
Tran oversees much of the work in Lake Mendota, from study design to data interpretation. She also plays a major role in the study of Lake Tanganyika. There, she is leading the analysis of nearly 500 genomes of microorganisms.
“The analyses consist of characterizing the whole microbial community of the lake,” says Tran. “We study their distribution patterns and abundances and use their genomes to identify their roles in carbon, nitrogen, and sulfur cycling.”
Anantharaman’s work — from hydrothermal vents to the human gut — hit a speed bump when the coronavirus pandemic closed labs in early 2020. While about half of what the lab does is computational and could continue remotely, the other half is experimental. There was little warning before labs were shuttered, and the bacteria and viruses Anantharaman’s team was growing slowly died off.
“There was a time where we completely shut down experiments, and that affected us very badly,” says Anantharaman. “It took us a good two months to get those experiments up and running again. Similarly, our collaborators were affected, and they had to prioritize their work.”
Even in late 2020, graduate students were only allowed to be in the lab for 20 hours per week, a less-than-ideal schedule for students trying to stay on top of coursework, conduct experiments, and calculate results.
What drives Anantharaman is knowing that full-scale and full-time research will return, as will trips to the bottom of the sea. He has National Science Foundation funding for another ride with Alvin in the spring of 2022. His team will conduct 18 submarine dives in collaboration with Samantha Joye of the University of Georgia and Roland Hatzenpichler from Montana State University.
“Unfortunately, on my previous trips, my graduate students could not go because they were taking classes,” Anantharaman says. “I’d definitely love to take my graduate students and postdocs on future trips.”
Such a trip would be a once-in-a-life-time chance for those graduate students: a chance to see the source of the samples they work with for hours in the lab, to witness the depths of the ocean where sulfur is cycling, and, on the way down, to take in a stellar lightshow seen nowhere else on earth.