Unveiling the Hidden Lives of Microbes Deep Underground

A group of scientists working at Bigelow Laboratory for Ocean Sciences have created a novel technique that connects the genetics and functionality of individual microbes that are oxygen-free and exist far below the surface of the Earth.

Understanding the function of microbial communities in global processes such as the carbon cycle requires an understanding of both of these attributes, which has long been a challenge in microbiology and requires connecting them.

The novel method, created at the Single Cell Genomics Center at Bigelow Laboratory, allowed scientists to find that, in a groundwater aquifer located nearly half a mile below the surface of Death Valley, one species of sulfate-consuming bacterium was both the most prevalent and the most active organism.

The results published in the Proceedings of the National Academy of Sciences, demonstrate the potential of this approach as a potent tool for determining the degree of activity of various organisms in these harsh settings.

Previously, we had to assume that all cells were operating at the same rate, but now we can see that there is a wide range of activity levels between individual members of the microbial communities. That helps us understand what these microbial communities are capable of and how that might influence global biogeochemical cycles.”

Melody Lindsay, Research Scientist and Study Lead Author, Bigelow Laboratory for Ocean Sciences

The new research is a component of a bigger effort that connects microbes’ genetic code, or the blueprint of their potential, to what those microbes are really doing at any given time.

The “Genomes to Phenomes” project, which is jointly sponsored by the University of New Hampshire, the Desert Research Institute, and Bigelow Laboratory, is funded by the NSF's EPSCoR program. It makes use of recent developments in single-cell genetic sequencing in a novel way by estimating the rates of processes occurring within those cells, like respiration, using flow cytometry.

Flow cytometry, a technique originally developed in the biomedical sciences, was adapted at Bigelow Laboratory to analyze individual environmental microbes. This method enabled researchers to isolate living microbes from aquifer water samples rapidly.

When specific chemical reactions occurred within the cell, these microbes were stained with a specially formulated compound designed to fluoresce under the flow cytometry laser. The correlation between the fluorescence intensity and the rate of these reactions was established experimentally using lab-grown cell cultures by student interns at Bigelow Laboratory, and this knowledge was then applied to the Death Valley samples.

The scientists sequenced each active cell's genome after measuring and separating them. Together with radioisotope tracers - a more conventional technique for gauging activity within a microbial community - the researchers also employed meta-transcriptomics to identify which genes are being actively expressed.

This was done to obtain additional information about the connections between these microbes' genetic potential and actual behavior, as well as to “double-check” the results.

The only analytical facility in the world providing researchers with this novel technique is the Single Cell Genomics Center.

This study was an exciting opportunity for our research team and the SCGC to help improve our understanding of the immense, enigmatic microbial ecosystems underground.”

Ramunas Stepanauskas, Senior Research Scientist, Principal Investigator, Bigelow Laboratory for Ocean Sciences

This study first demonstrated The approach for measuring individual cells' activity. A small percentage of microorganisms in the ocean are responsible for consuming the majority of the oxygen, according to research the team published in late 2022 on microbes in seawater.

The team is expanding that method in this latest study to demonstrate that the approach can be applied in low biomass environments containing oxygen-free microbes.

As an illustration, the scientists calculated that the subsurface aquifer samples from California contained hundreds of cells per ml of water instead of millions of cells in ml of surface water on average.

We started out with oxygen-respiring organisms in the ocean because they’re a little more active, a little easier to sort, and easier to grow in the lab. But aerobic respiration is just one process that is possible in microbiology, so we wanted to branch out beyond that.”

Melody Lindsay, Research Scientist and Study Lead Author, Bigelow Laboratory for Ocean Sciences

The findings demonstrated that the most prevalent and active microbe in this setting, reducing sulfate for energy, was Candidatus Desulforudis audaxviator.

Though there were significant variations in the individual microbes' levels of activity, the team’s measured overall activity rates were lower than those of the seawater samples from the earlier investigation.

The research group is currently working on adapting their technique to new settings, such as the sediments along Maine’s coast, and to measure other anaerobic reactions, like nitrate reduction. Lindsay and colleagues are also able to test the technique related to a NASA-funded project deep below the ocean’s surface.

Lindsay added, “Right now, we’re getting all of these point measurements around the world, and they do help us better understand what microbes are up to, but we need to scale it up. So, we’re thinking about how to apply this method in new places, even potentially on other planets, in expanded ways.”