A New Tool for Investigating Microbial Methane Production in Natural Environments

Isocyanides enable detection of nickel-free methane production in environmental samples

Methane-producing microbes play a central role in the global carbon cycle and contribute significantly to the greenhouse effect. In 2025, researchers led by Dr. Seigo Shima and Dr. Shunsuke Nomura at the Max Planck Institute for Terrestrial Microbiology demonstrated that under severe nickel deficiency—as present in many natural habitats—methanogens employ alternative metabolic pathways. Instead of nickel-containing enzymes, they use a nickel-free enzyme called [Fe]-hydrogenase (Hmd). For the first time, the researchers found that certain organic compounds, isocyanides, specifically inactivate this nickel-free enzyme even in natural samples. This discovery enables a straightforward method to determine whether and to what extent this alternative methane formation occurs in nature.

Methanogenic Archaea are microorganisms that can produce methane (CH₄) from carbon dioxide (CO₂) and hydrogen (H₂), a process contributing substantially to global greenhouse gas emissions. It has been long accepted that these microorganisms require nickel to generate energy, as they utilize specialized enzymes -for example [NiFe] hydrogenases - to activate hydrogen and facilitate electron transfer during methane production.

In 2025, researchers led by Dr. Seigo Shima and Dr. Shunsuke Nomura at the Max Planck Institute for Terrestrial Microbiology demonstrated that under severe nickel limitation — common in many natural environments — methanogens utilize alternative metabolic pathways (Link to Press Article). Instead of depending on nickel-containing enzymes, they employ a nickel-free enzyme, [Fe] hydrogenase (Hmd), which uses iron to transfer hydrogen directly to the substrate. This finding represents a paradigm shift by disproving the long-held assumption that only [NiFe] hydrogenases are functional in the metabolism. Under natural conditions, Hmd may serve as the primary electron transfer agent in methane metabolism, thus acting as a key regulator of natural methanogenesis. 

A chemical “switch” reveals the reaction in nature

Building on these findings, the researchers developed a tool for further investigation. It is well established that isocyanides inhibit the Hmd enzyme with high affinity. The study examined whether isocyanides could serve as targeted “switches” to detect methane production via the nickel-free pathway in natural samples. To this end, the researchers tested isocyanides in two systems. In cell extracts from methanogens cultured under nickel-deficient conditions, methane production was strongly inhibited by isocyanides. A similar inhibition pattern appeared in living cell cultures (cell suspensions), especially with hydrophobic isocyanides such as 2-naphthyl isocyanide (NIC), which penetrate cells more effectively.

“The inhibition of methane production exclusively in cells grown under nickel-deficient conditions is a clear indicator,” states Dr. Shunsuke Nomura. “If methane production decreases sharply after isocyanide treatment, it strongly suggests that the Hmd-dependent pathway is the major system.” Until now, directly assessing the occurrence and extent of nickel-free methanogenesis in environmental samples has been challenging. Techniques like metaproteomics or metatranscriptomics are costly, complex, and require specialized laboratories. In contrast, the new isocyanide-based method is simple, cost-effective, and suitable for future field studies involving samples from ocean floors, peatlands, or soils.

“Isocyanides selectively inhibit the nickel-free pathway without affecting the classical pathway,” explains Dr. Seigo Shima. “This specificity makes them an ideal tool to quantify the contribution of Hmd-dependent methane production in nature.”

A Tool for Climate Research

The discovery that isocyanides specifically inhibit Hmd under natural conditions opens new avenues for understanding global methane production. Determining the proportion of this nickel-free pathway in natural ecosystems enables more accurate assessment of how changes in nutrient availability—caused by climate change or human interventions—affect methane emissions.

Additionally, the method may help identify microbes particularly adapted to nutrient-poor habitats. In the future, it could also support research on potential countermeasures, such as reducing methane emissions in agricultural systems or wastewater treatment plants.