A team of scientists led by researchers at the Bigelow Laboratory for Ocean Sciences has developed an innovative method that connects the genetics and functioning of individual microbes living without oxygen deep below the Earth’s surface. Measuring both of these properties and, more importantly, correlating them has long been a challenge in microbiology but is critical to understanding the role of microbial communities in global processes such as the carbon cycle.

The new approach, developed at the Bigelow Laboratory Center for Single Cell Genomics, allowed researchers to discover that a sulfate-consuming bacterial species was not only the most abundant but also the most active organism in the groundwater aquifer beneath Death Valley. half a mile down. surface. The results were published on: Proceedings of the National Academy of Sciences shows how this method can be a powerful tool for measuring the activity of different organisms in these harsh environments.

Insights into the dynamics of the microbial community

“Previously we had to assume that all cells were working at the same rate, but now we see that there is a wide range of activity levels among individual members of microbial communities,” said researcher and lead author Melody. Lindsay. “This helps us understand what microbial communities are capable of and how this may affect global biogeochemical cycles.”

The latest research is part of a larger project that links microbes’ genetic code (the blueprint for what they can do) to what they actually do at any given moment.

Methodological achievements

The Genomes to Events Project, funded by NSF’s EPSCoR program, is a joint venture between the Bigelow Laboratory, the Desert Research Institute, and the University of New Hampshire. It uses recent advances in single-cell genetic sequencing with a creative approach to the application of flow cytometry to assess the rate of processes such as respiration occurring in these cells.

Flow cytometry, a method for analyzing individual environmental microbes adapted at the Bigelow Biomedical Sciences Laboratory, allowed researchers to quickly isolate live microbes in aquifer water samples. These microbes were stained with a specially designed compound that glows under a flow cytometry laser when certain chemical reactions occur in the cell. The relationship between the intensity of the cells’ laser-induced fluorescence and the rate of these reactions was studied experimentally in laboratory-grown cell cultures by student interns in the Bigelow laboratory and then applied to samples taken from Death Valley.

Once the active cells were measured and isolated, the team sequenced their individual genomes. The researchers also used meta-transcriptomics, a method for determining which genes are actively expressed, and radioisotope tracers, a more traditional method for measuring activity in a microbial community. This was done both to “double-check” the results and to learn more about the connections between what these microbes can do genetically and what they actually do.

The Center for Single Cell Genomics is the only analytical center in the world offering this new technique to researchers.

“This study is a great opportunity for our research team and the SCGC to help advance our understanding of the vast, mysterious microbial ecosystems underground,” said Bigelow Laboratory Senior Scientist Ramunas Stepanauskas, SCGC director and principal investigator of the project.

This new study builds on the first demonstration of this approach to measure the activity of single cells. The team published results from a study of microbes in seawater in late 2022; This showed that a small proportion of microorganisms were responsible for consuming most of the oxygen in the ocean. In this new paper, the team extends this method to show that it can be used in low-biomass environments with non-oxygen-dependent microbes. For example, in samples taken from an underground aquifer in California, scientists estimated that there were hundreds of cells per milliliter of water, compared to millions of cells in a typical milliliter of surface water.

“We started with oxygen-breathing organisms from the ocean because they’re a little more active, easier to isolate, and easier to grow in the lab,” Lindsay said. said. “But aerobic respiration is only one process possible in microbiology, so we wanted to go beyond that.”

Expanding the scope of microbial research

The results confirmed the presence of bacteria Applicant Desulforudis audaxviator was not only the most abundant microbe in this environment, but also the most active microbe in reducing sulfate for energy. The overall activity rates the team measured were low compared to seawater samples from a previous study, but there were large differences between the activities of individual microbes.

The research team is currently working to apply their method to measure other anaerobic reactions, such as nitrate reduction, and to new environments, including sediments along the Maine coast. A related NASA-funded project also gives Lindsay and his colleagues the opportunity to test the method deep in the ocean.

“We’re doing all these spot measurements around the world now, and they’re really helping us better understand what microbes are up to, but we need to scale up,” Lindsay said. “So we are thinking about how to apply this method in expanded ways to new places, potentially even to other planets.”