Billions of tons of greenhouse gases are trapped under the sea, and that's a good thing. Along the continent's coast, which slopes down into the ocean, tiny cages of ice hold methane gas in place, preventing it from rising and being released into the atmosphere. Although not often highlighted in the media, these formations, known as methane clathrates, have come under scrutiny for their possible impact on climate change. In offshore drilling operations, methane ice can clog pipes, causing them to freeze and rupture. The 2010 Deepwater Horizon oil disaster is suspected to have been caused by the accumulation of methane clathrates.
Methane clathrates (white ice-like material) beneath seafloor rocks in the northern Gulf of Mexico. Such deposits indicate that methane and other gases travel across the seafloor and into the ocean. Image source: NOAA
But until now, the biological processes of how methane gas remains stable on the seafloor were almost completely unknown. In a groundbreaking study, an interdisciplinary team of Georgia Tech researchers has discovered a previously unknown class of bacterial proteins that play a critical role in the formation and stability of methane inclusion complexes.
The research team, led by Jennifer Glass, associate professor in the School of Earth and Atmospheric Sciences, and Raquel Lieberman, professor in the School of Chemistry and Biochemistry and Sepcic-Pfeil Chair, showed that these novel bacterial proteins are as effective in inhibiting the growth of methane clathrates as commercial chemicals currently used in drilling wells, but are nontoxic, environmentally friendly, and scalable. Their research, funded by NASA, informs the search for life in the solar system and could also improve the safety of natural gas transportation.
The study, published in the journal PNAS Nexus, highlights the importance of basic science in studying Earth's natural biological systems and highlights the benefits of interdisciplinary collaboration.
"We wanted to understand how these formations remain stable on the seafloor and what exactly are the mechanisms that contribute to their stability," Glass said. "This is something no one has done before."
Screening sediment
The work began with the team examining samples of clay-like sediment that Glass collected from the seafloor off the Oregon coast.
Glass hypothesizes that the sediment contains proteins that influence the growth of methane clathrates, similar to well-known antifreeze proteins in fish that help them survive in cold environments.
Morphological effects of inhibitors on methane cage shells. Left: A cartoon showing the formation of methane inclusion complexes at the onset of inclusion growth and at 3 hours with and without inhibitors. Right: Representative photographs of experimental methane clathrates at each growth stage, labeled by treatment. Image source: Georgia Tech
But to confirm her hypothesis, Glass and her research team first had to identify candidate proteins among the millions of potential targets contained in the sediment. They then needed to make the proteins in the lab, despite not understanding how those proteins behaved. Moreover, no one had studied these proteins before.
Glass approached Lieberman, whose lab studied the structure of proteins. The first step is to use DNA sequencing combined with bioinformatics to identify the genes for proteins contained in the sediment. Dustin Huard, a researcher in Lieberman's lab and first author on the paper, then prepared candidate proteins that might bind to the methane inclusion complex. Huard uses X-ray crystallography to determine the structure of proteins.
Creating seafloor conditions in the laboratory
Huard gave the candidate protein to former doctoral student Abigail Johnson. student in Glass' lab and co-first author of the paper, now a postdoctoral researcher at the University of Georgia. To test these proteins, Johnson recreated the high pressures and low temperatures of the ocean floor in the lab, forming methane clathrates himself. Johnson worked with Dai Sheng, an associate professor in the School of Civil and Environmental Engineering, to build a unique pressure chamber from scratch.
Johnson placed the protein into a pressure vessel and adjusted the system to simulate the pressure and temperature conditions required for inclusion complex formation. By pressurizing the vessel with methane, Johnson forced the methane into the droplets, forming a methane clathrate structure.
She then measured the amount of gas consumed by the clathrates—a measure of how quickly and how much clathrates formed—in the presence and absence of protein. Johnson found that using clathrate-binding proteins, less gas was consumed and the clathrate compounds melted at higher temperatures.
When the research team confirmed that these proteins affected the formation and stability of methane inclusion complexes, they conducted molecular dynamics simulations using Huard's protein crystal structure with the help of James (JC) Gumbart, a professor in the School of Physics. The simulations allowed the team to identify the specific sites where the protein binds to the methane inclusion complex.
A surprisingly novel system
The study revealed unexpected insights into protein structure and function. The researchers initially thought that a portion of the protein similar to fish antifreeze proteins would play a role in inclusion complex binding. Surprisingly, this part of the protein plays no role and an entirely different mechanism directs the interaction.
They found that these proteins do not bind to the ice but interact with the inclusion structure itself, directing its growth. Specifically, parts of the protein that have similar properties to antifreeze proteins are buried in the protein structure and instead serve to stabilize the protein.
The researchers found that these proteins performed better at modifying methane clathrates than any antifreeze protein tested in the past. They perform just as well, if not better, than the toxic commercial inclusion complex inhibitors currently used in drilling, which pose a serious threat to the environment.
Preventing clathrate formation in natural gas pipelines is a multi-billion dollar industry. If these biodegradable proteins could be used to prevent catastrophic natural gas leaks, the risk of environmental damage would be greatly reduced.
"We were lucky that this actually worked because although we selected these proteins based on their similarity to antifreeze proteins, they are completely different," Johnson said. "They have similar functions in nature but do it through completely different biological systems, which I think is really exciting."
Methane clathrates may exist throughout the solar system—for example, in the subsurface of Mars, and on icy moons in the outer solar system, such as Europa. The team's findings suggest that if microbes existed on other planetary bodies, they might produce similar biomolecules to retain liquid water in the channels of clathrates, thereby supporting life.
"We still know a lot about the fundamental systems on Earth," Huard said. "That's one of the great things about Georgia Tech - different communities can come together to do really cool, unexpected science. I never thought I'd be working on an astrobiology program, but here we are, and we're very successful."