Researchers at the University of California, Berkeley have published a study in Science that explores how the activity of a key microbial enzyme influences the isotopic composition of methane, complicating efforts to trace environmental sources of this greenhouse gas. Methane is responsible for a significant portion of global warming, with about two-thirds of its emissions coming from microbes living in oxygen-free environments such as wetlands, rice fields, landfills, and animal digestive systems.

Tracking methane’s origins has been more difficult than tracking carbon dioxide because it requires analyzing the isotopic fingerprints—specific ratios of carbon and hydrogen isotopes—in methane molecules. The new research focuses on methanogens, microorganisms that produce methane as they break down organic matter in low-oxygen environments.

“When we integrate all the sources and sinks of carbon dioxide into the atmosphere, we kind of get the number that we’re expecting from direct measurement in the atmosphere. But for methane, large uncertainties exist — within tens of percents for some sources — that challenge our ability to precisely quantify the relative importance and changes in time of the sources,” said UC Berkeley postdoctoral fellow Jonathan Gropp, who is first author of the paper. “To quantify the actual sources of methane, you need to really understand the isotopic processes involved in producing the methane.”

Gropp collaborated with molecular biologist Dipti Nayak and geochemist Daniel Stolper at UC Berkeley to use CRISPR gene editing technology to adjust levels of methyl-coenzyme M reductase (MCR), an enzyme essential for methane production by methanogens. This allowed them to observe how changes in enzyme activity affect both microbial metabolism and resulting isotope signatures.

“It is well understood that methane levels are rising, but there is a lot of disagreement on the underlying cause,” said co-author Dipti Nayak, UC Berkeley assistant professor of molecular and cell biology. “This study is the first time the disciplines of molecular biology and isotope biogeochemistry have been fused to provide better constraints on how the biology of methanogens controls the isotopic composition of methane.”

Many elements occur naturally as mixtures of heavier or lighter versions called isotopes. The distribution pattern—or fingerprint—of these isotopes can be used to trace biological processes. Scientists have traditionally assumed that differences in these fingerprints depend mainly on what type of food source methanogens consume.

“Over the last 70 years, people have shown that methane produced by different organisms and other processes can have distinctive isotopic fingerprints,” said geochemist and co-author Daniel Stolper, UC Berkeley associate professor of earth and planetary science. “Natural gas from oil deposits often looks one way. Methane made by the methanogens within cow guts looks another way. Methane made in deep sea sediments by microorganisms has a different fingerprint. Methanogens can consume or ‘eat,’ if you will, a variety of compounds including methanol, acetate or hydrogen; make methane; and generate energy from the process. Scientists have commonly assumed that the isotopic fingerprint depends on what the organisms are eating, which often varies from environment to environment, creating our ability to link isotopes to methane origins.”

The researchers found that not only does diet matter but so do environmental conditions and how microbes respond at a genetic level.

“I think what’s unique about the paper is, we learned that the isotopic composition of microbial methane isn’t just based on what methanogens eat,” Nayak said. “What you ‘eat’ matters, of course, but the amount of these substrates and the environmental conditions matter too, and perhaps more importantly, how microbes react to those changes.”

“Microbes respond to the environment by manipulating their gene expression, and then the isotopic compositions change as well,” Gropp said. “This should cause us to think more carefully when we analyze data from the environment.”

Their experiments showed that when MCR enzyme levels are reduced—mimicking nutrient scarcity—the cells alter many other enzymes’ activities as well. This leads to slower rates of methane generation where certain reactions run both forwards and backwards inside cells. As a result, hydrogens incorporated into newly formed methane increasingly reflect those found in water rather than solely those from food sources like acetate or methanol.

“This isotope exchange we found changes the fingerprint of methane generated by acetate and methanol consuming methanogens vs. that typically assumed. Given this, it might be that we have underestimated the contribution of acetate-consuming microbes, and they might be even more dominant than we have thought,” Gropp said. “We’re proposing that we at least should consider the cellular response of methanogens to their environment when studying isotopic composition of methane.”

The CRISPR technique developed could also help scientists manipulate other metabolic pathways in archaea for future studies related to Earth’s history or even practical applications such as reducing harmful emissions or redirecting microbial energy toward useful products.

“This opens up a pathway where modern molecular biology is married with isotope-geochemistry to answer environmental problems,” Stolper said. “There are an enormous number of isotopic systems associated with biology and biochemistry that are studied in the environment; I hope we can start looking at them in the way molecular biologists now are looking at these problems in people and other organisms — by controlling gene expression and looking at how stable isotopes respond.”

For Nayak’s team this could mean eventually engineering microbes so they produce less environmentally damaging gases: “By reducing amount this enzyme makes methane…we can essentially give them another release valve…to put those electrons…into something else…more useful,” she said.

Other contributors include Markus Bill (Lawrence Berkeley National Laboratory), Rebekah Stein (former UC Berkeley postdoc), Max Lloyd (Penn State University). Funding came from organizations such as Alfred B. Sloan Research Fellowships; Nayak also works with Chan-Zuckerberg Biohub.

The full study appears online: Modulation of methyl-coenzyme M reductase expression alters isotope composition.