Amino Acid Metabolism Fuels Fracking Communities

A small and interconnected microbial community lives in deep shales. The microbes persist in the salty fluid-filled cracks created by hydraulic fracturing. They survive by consuming amino acids such as glycine betaine, a compound that protects the microbes from the high-salt environment. Its metabolic byproducts also provide the raw ingredients for making methane.

Hydraulic fracturing, or “fracking,” has enabled natural gas companies to reach new heights in natural gas production. By pumping a mix of water and chemical additives into shale deposits, companies break up the rock to release more gas. This process opens up a subsurface habitat, seeding it with microbes carried along in the fracking fluid. A handful of organisms survive the high temperature, pressure, and salinity of their new home, 2,500 meters beneath the surface.

Fracking rig
Researchers were on hand during this early-morning vertical drilling into the Marcellus shale in West Virginia. Credit: Rebecca Daly

In a new paper in the journal Proceedings of the National Academy of Sciences [1], a large collaboration including DCO members Kelly Wrighton, Michael Wilkins (both at Colorado State University, USA), David Cole, Julie Sheets (both at The Ohio State University, USA), Timothy Carr (West Virginia University, USA), and Paula Mouser (University of New Hampshire, USA), examines the community of microbes that persist in these environments, and tease apart the ways that they compete, cooperate, and prey on each other. Using techniques to catalogue all of the genomes and metabolites in methane well fluids, the researchers show that the community survives by fermenting amino acids and their derivatives, especially glycine betaine. 

Many microbes build up stores of glycine betaine in the cell to protect themselves from the high salinity that occurs when fracking fluids dissolve underground salt deposits. But this compound serves a dual purpose. Microbes use it for energy and growth. “The microbes are producing glycine betaine and then it’s sustaining the ecosystem,” said Wrighton. “It’s almost like a ‘perpetual energy machine.’”

This project began when Rebecca Daly and Mikayla Borton (in the Wrighton laboratory, formerly at The Ohio State University) sequenced microbial DNA from multiple gas wells across Appalachia and saw the same handful of species in each well [2], despite differing locations and fracking fluids. Through funding from the DCO Census of Deep Life, they sequenced the metagenomes from five samples in one well. “The support for the metagenomes was critical in getting this project off the ground,” said Wrighton. Once the researchers knew the identity of the microbes, they could investigate the metabolisms that keep them alive in such challenging environments. 

From their previous work, they knew that cells from these communities had the necessary genes for metabolizing glycine betaine, but they didn’t know what was driving the process. Microbes can convert glycine betaine into ingredients for methanogenesis but they need another amino acid to complete the reaction and generate energy for the cell. 

Mikayla Borton
Lead author Mikayla Borton enjoys her time collecting samples in the field. Credit: Rebecca Daly

To bring those microbial communities into the lab, where they could be studied more easily, the researchers cultured produced fluids from shale wells in bottles. They added glycine betaine to some bottles, but not to others. When they sequenced the organisms in the cultures, at first they were disappointed to see that both sets had highly similar communities, with or without added glycine betaine. But then they realized that since the microbes produce their own glycine betaine to protect themselves from the salty fluids, adding extra just resulted in the same organisms, but more of them.

Four types of microbes thrived inside the cultures, using highly interconnected metabolisms. Some microbes gain energy by metabolizing glycine betaine using another amino acid, sarcosine. This reaction yields compounds with methyl groups that some methanogens convert into methane. Glycine betaine is valuable and expensive for cells to produce, but it goes up for grabs when viruses infect and burst the microbes. The researchers even detected evidence that viruses were actively preying on microbes in the bottles.

To see if the same metabolisms in their cultures occurred in fracked shale deposits, the researchers collected 40 fluids samples from five wells in the Utica and Marcellus shale deposits. They sequenced the metagenomes and identified all the metabolites in the fluids, called the metabolome, and compared them to the geochemistry of the fluids. “We found that those metabolites that were most important across wells were glycine betaine and amino acids. We could use the organismal abundance to predict those metabolites,” said Borton, a lead author on the recent study. 

Rebecca Daly (left) and Julie Sheets (right)
Rebecca Daly (left) and Julie Sheets (right), worked together to collect biological and geochemical data. Credit: Susan Welch

The geochemical analysis of the fluids, performed by Susan Welch (The Ohio State University) and David Cole indicated that the salinity of each well, which impacts glycine betaine production, is the main factor affecting the composition of the community.

Understanding which microbes eat specific compounds could be useful for bumping up methane production by specifically stimulating methanogens with their preferred compounds. Some companies pump acetate into coal-bed methane wells because it stimulates the methanogens in these regions. 

Many studies of carbon cycling in microbial communities overlook the role of amino acids and other organic nitrogen compounds because the software tools to identify the required genes within a data set are limited, and we lack ability to track how the metabolites move through a community. “These metabolisms are really pervasive across ecosystems,” said Wrighton. “Organic nitrogen cycling is an important component of the carbon cycle that we’ve largely ignored.” Next, Borton and Wrighton will look into similar metabolisms in the human gut, where the same metabolites are linked to atherosclerosis. But they suspect that these processes are also active in soil, and numerous other ecosystems.

Wrighton and her lab group just relocated to Colorado State University and they plan to add samples from Colorado shale methane wells into their analyses. They also are sampling from wells in Texas, Oklahoma, and North Dakota, to see how the community and metabolisms compare to wells in Appalachia.

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