Bringing Microbial Dark Matter Into the Light
When Belinda Ferrari, Ph.D., spent 2 1/2 weeks in Antarctica, she didn't know what to expect. Staying in a hut barely big enough for the whole team was not on her radar—neither was birds flying away with collection tubes. Ferrari, a professor of biotechnology and environmental microbiology at University of New South Wales, was in Antarctica hunting for microbes. She was collecting soil samples that, to the naked eye, looked devoid of life. But looks can be deceiving: the soil was, in fact, home to a wealth of previously undiscovered bacteria.
Antarctic soil is only 1 example of the microbial goldmine hiding in "extreme" environments. Hot springs, deep subsurfaces, caves and volcanoes are just a few of the places where microbiologists have found an abundance of unexplored and uncultured microbes. In the unknown lies a plethora of potential; previously uncharacterized microbes can have applications in bioremediation, human health and antibiotic discovery.
Microbiology hasn't always ventured into the wild like this. Robert Koch, often called the father of modern microbiology, first grew microbes in pure culture in 1884. "Once we had the first pure culture, microbiology focused fairly heavily on pure cultures, and for good reason," said Brian Hedlund, Ph.D., a professor of life sciences at University of Nevada, Las Vegas. Pure cultures can be genetically tractable, help link specific microbes to a disease and are less "messy" than studying a microbe alongside its friends.
While microbiologists have relied on Petri dishes to study microbes in the last century, most of Earth's microbial diversity hasn't been grown in the lab before. This "microbial dark matter" exists in our eyes as DNA sequences or microscopy images that hold clues to how mysterious microbes live. Microbiologists are now using such clues as jumping-off points to begin culturing the once unculturable.
How Sequencing Exposes the Magnitude of Microbial Dark Matter
The rise of DNA sequencing has opened a new world for microbiologists, offering a glimpse of just how much microbiology remains unknown. "It's totally new, uncharted territory," said Karen Lloyd, Ph.D., a professor of earth sciences and of marine and environmental biology at the University of Southern California, who studies novel microbes in hot springs. "[There’re] things that we really wouldn't know exist to any degree if we didn't have environmental DNA sequencing."
First came amplicon sequencing, which gave microbiologists a way to see what bacteria are in a sample by looking only at the 16S rRNA. Metagenomics, shotgun sequencing and long read sequencing came next, providing a broader view into a microbe's entire genome. With these sequences, microbiologists could assemble genomes and begin to understand the broader functional capabilities of individual microbes.
Metagenomic sequencing was pivotal to what Lloyd called one of the "really big discoveries" in microbial dark matter research: the discovery of the Asgard archaea in marine sediments near Loki's Castle, a hydrothermal vent in the mid-Atlantic Ocean, in 2015. These archaea lie on the edge between eukaryotes and prokaryotes. Studying these microbes can help microbiologists understand how early microbial cells became larger with more defined organelles, leading to complex multicellular life, Lloyd explained.
These sequencing efforts have made microbiologists realize just how much microbiology cannot be confined to the laboratory. In 2018, Lloyd used metagenomics and metatranscriptomics in a project that she called "a real labor of love" to estimate that 81% of microbial cells are uncultured genera and 25% are uncultured phyla.
Doing Microbiology Without Pure Cultures
Beyond sequencing, there are still many ways to understand microbes without growing them. The candidate bacterial phylum Omnitrophota sat largely unexplored for 25 years after microbiologists found their 16S rRNA sequences in a Yellowstone hot spring. These microbes—whose sequences have since been uncovered in geothermal environments, freshwater lakes and springs—were characterized in depth in 2023, when Hedlund began studying their biology. Fluorescence-activated cell sorting helped his team estimate cell diameters to show that the bacteria are ultrasmall nanobacteria, about 100-250 times smaller than E. coli by volume. Genomic analysis hinted that these tiny bacteria were possibly predatory or parasitic, as the bacteria contained gene clusters typically found in bacterial symbionts.
Bacteria with predatory and parasitic lifestyles generally have higher metabolic rates, so Hedlund's team wanted to see if this was the case for Omnitrophota bacteria. The team turned to quantitative stable isotope probing (qSIP), which labels newly synthesized DNA with heavy isotopes, as a reporter for metabolic activity. The team used existing qSIP data from soil samples and found that Omnitrophota bacteria incorporated these isotopes into their DNA at higher rates than non-predatory bacteria, pointing to robust metabolic activity and, likely, a predatory lifestyle.
Lloyd, who noted that much of microbial dark matter research is inference, took another culture-independent approach with samples collected from 50 meters below the surface of the Baltic Sea. She studies microbes from this region that take thousands of years to double but can remain metabolically active. However, microbiologists didn't know how these microbes survive and function in their environments. Lloyd's team combined a multiomic approach with enzyme assays to decipher the subsistence mechanisms of these microbes. The team was able to pinpoint differences in what these microbes metabolize based on enzyme activities and transcript abundances, demonstrating how microbiologists can still learn a lot about microbes without growing them in the lab. "[This] was really thrilling for me. Because we're doing so much inference, we often have to measure things a bunch of different ways" in order to have more confidence in the findings, she explained. "To see [these methods] match up so well was really huge."
Using Sequence Data to Guide Culturing Strategies
The lack of pure cultures hasn't stopped microbiologists; they've uncovered a lot using culture-independent methods, particularly when it comes to how microbes behave in their natural environments. "The notion that a glass container with a single organism without its friends would survive seems kind of unreasonable," said Lloyd, who prefers to study how an organism acts in nature. "Nature is messy and weird and uncontrolled. I will never perfectly replicate natural conditions in my laboratory."
But despite the difference between the lab and the natural environment, there's still a lot to gain from studying cultivated microbes. Scientists often see these contrasting approaches as complementary. "The culture-independent methods tell you who's there and what they can potentially be doing," Ferrari said. "But unless you get an isolate, you can't actually test some of these hypotheses that you're building."
Culture-independent tools act as guideposts for cultivation. "You should use metagenomic data to choose the isolation or enrichment strategy. Cultivation should really not be done without multiomic data," Hedlund explained. Culturing microbial dark matter is no easy task, however. It can take years, and there's no guarantee of success. For Ferrari, "it's [about] trying as many out-of-the-box ideas you can think of ... [the microbes] are not going to grow on standard media, so [you're] trying to think about other ways to get these organisms to grow."
While in Antarctica, Ferrari identified 2 candidate phyla, WPS-2 and AD3, in soil samples. "We only knew of them from 16S sequences. That's all we knew about them," she said. Yet, those bacteria made up more than 30% of the community in the samples. Following up with shotgun sequencing, Ferrari got the first genomes from these phyla. The genomes contained genes for high-affinity hydrogenases and carbon monoxide dehydrogenases, which presumably help the microbes consume trace gases from the atmosphere. "Because they consume a lot of hydrogen, when we set up [enrichment cultures], we have very low concentrations of nutrients in the media," she said. "Then we add excess hydrogen in the headspace. We think that's the energy source."
The team has had enrichment cultures of WPS-2 and AD3 going for 6 years, and they were just finally able to culture WPS-2 on a plate. Ferrari recalls her student coming to her shaking with the news. Culturing bacteria on plates opens new avenues of research, such as for experiments relying on single clones or spatial gradients.
This genomics-informed approach to cultivation has accelerated in recent years, largely driven by the sheer amount of data collected from omics and a variety of culture-independent tools that can help inform growth strategies. Morten Sommer, Ph.D., a professor of bacterial synthetic biology at Technical University of Denmark, used metagenomes to guide the inclusion of medium additives for targeted enrichment of gut microbes, finding links between medium preferences and phylogeny. Another study from the group of Nikolai Ravin, Ph.D., a professor of bioengineering at the Russian Academy of Sciences, used metagenome-assembled genomes to inform the cultivation of thermophilic spirochetes, resulting in 2 new pure culture strains.
The Future of Studying Microbial Dark Matter
With the vast amount of information sequencing and single-cell techniques generate, Hedlund is excited about combining AI and robotics to transform data into experimental strategies. For example, AI can make predictions about the best media and growth conditions for a certain microbe and automation can test out these conditions in parallel using microtiter plates. Microbiologists are beginning to do this. Harris Wang, Ph.D., chair and professor of systems biology at Columbia University, has built a high-throughput machine learning and robotic strain isolation platform to isolate microbes from gut microbiome samples. Another team, led by Paul Jensen, Ph.D., an associate professor of biomedical engineering at the University of Michigan, developed an automated AI platform to predict needs for microbial metabolism with no prior data.
"You don't need a 100 mL culture anymore to do good experiments," Hedlund said. "And you don't need a pure culture to do experiments."
Still, microbiologists are continuing to uncover the scale of Earth's uncultured life. "We can see the tip of the iceberg," said Lloyd. "What we don't know is almost certainly orders of magnitude more than what we do know."
What's next in the world of microbial dark matter research? ASM Microbe 2026 is the place to find out. Join us in Washington, D.C., June 4-7 to share your research and connect with scientists uncovering the processes driving microbial functions and interactions.
Author: Jennifer Tsang, Ph.D.
In This Issue
- Why Does Basic Science Research Matter?
- The Value of Curiosity-Driven Research: Mechanism Discovery With Glen McGugan
- Stuck on You: How Bacteria Migrate and Adhere to Their Hosts
- Cable Bacteria: Electric Marvels of the Microbial World
- Can't Sleep? Your Microbiome May Play a Role
- How Spatial Structure Shapes Microbial Communities
- Plasmids and the Spread of Antibiotic Resistance Genes
- From Mpox to Measles: Why Viruses Cause Lesions and Rashes
- Bringing Microbial Dark Matter Into the Light