Sometimes, the best way to study a community of living things is by looking at their leftovers. And just as wildlife biologists study scat to learn what a pack of predators is eating—a team of student microbiologists at CU Boulder is looking at the dark, slimy sewers beneath the university’s campus to learn more about what the people are doing above.

“Sewage systems are a unique ecosystem we don’t always think about: an underground river network connecting buildings,” said Noah Fierer, CIRES Fellow, professor of Ecology and Evolutionary Biology at CU Boulder, and director of CU’s Center for Microbial Exploration. “Studying the microbial communities in sewage allows us to monitor human populations unobtrusively.”

During CU’s fall semester, a group of PhD students played the part of microbial treasure hunters. During the student-led graduate course, the team investigated sewage samples in an open-ended, ‘choose-your-own adventure’ biology study. The group poured through sequenced DNA from sewage sludge, looking for patterns in the complex collage of human, plant, bacterial and viral genetic material—and hope to publish their findings later this year.

Sewage research can uncover changes in people’s diets, trends of viral transmission, or even antibiotic resistance patterns in bacteria, the team says. “You get waste matter from toilets, outflow from showers and sinks, and even organisms and chemicals from outdoor soil that wash into the system,” said Fierer.

Just a couple weeks earlier, Cresten Mansfeldt, CU Boulder Environmental Engineering assistant professor, knelt over a manhole cover near a CU Boulder East Campus dorm, pulling up water from a collection tube threaded deep into the underground flow of sewage. Last fall, Mansfeldt and his colleagues pulled up samples daily from 20 locations across CU Boulder campus, to look for COVID-19 in the wastewater.

Fierer saw an opportunity to piggyback on Mansfeldt’s efforts. The team could also use the samples to investigate the mess of microbes that live in sewage systems, to learn more about how these communities behave and change over time and with building occupancy.

“There’s a lot we can learn about human ecology if we look to the sewers,” said Emily Kibby, a CU Boulder Biochemistry PhD student who participated on the project.

The students dove into the samples with open minds: they didn’t know what they’d find. The team started by poring over the collection of DNA sequences extracted from the sewage samples and then developed research questions as patterns in the samples revealed themselves.

Viruses

And in Kibby’s case, she and her research group investigated viruses. The team saw a relationship between location and viral community structure—sewage from different buildings harbored distinct viral communities. This pattern is a product of associated changes in the bacterial communities as most of the viruses detected were predators of bacteria, highlighting the close links between bacteria and the viruses that infect them, the team says.

“Sewage microbes, including viruses which we studied, are still largely underexplored,” said Corinne Walsh, a CU Boulder EBIO and CIRES PhD student in the same working group. “There’s so much left to learn.” Walsh calls the sewage samples the team studied “grab bags”—each with its own complex and fascinating mix of microscopic information, with endless possibilities to explore.

Bacteria and More

And viruses weren’t the only thing in that sewage grab bag. John Sterrett, a CU Boulder Integrated Physiology PhD student, worked with his group to investigate bacteria.

Sterrett and his team observed bacterial communities before and after a massive snow storm that dumped several inches of meltwater into the sewers in September 2020. The precipitation event transformed the amounts and types of bacteria found in sewage, most likely due to increased input of soil bacteria into the sewage system after the snow event, the team says.

Sterrett’s team also looked at those bacteria commonly found in human feces. Intuition may suggest that the more people use toilets in a building, the more fecal bacteria might be present, Sterrett said. However, this was not the only way human activity affected the bacterial communities. In fact, the lack of human activity during COVID-19 shutdowns resulted in  stagnant water which served as a place for certain bacteria to multiply. More people means higher volumes of water moving through sewer systems that can effectively change the bacterial communities found there.

“This work reveals how much human occupancy and behavior can impact what ends up living in our sewers,” said Sterrett. "In this case, the COVID-19 shutdowns posed a unique issue: some pathogens could flourish in the stagnant water from the atypical lack of human activity in the building."