Unlocking the Mysteries of Biological Nitrogen Fixation: The Role of Mineral-Bound Trace Metals

Nitrogen is an essential element for life, serving as a building block for amino acids, proteins, and nucleic acids. However, the gaseous form of nitrogen (N₂) that dominates our atmosphere is notoriously unreactive, rendering it inaccessible to most organisms. This challenge has long puzzled scientists, leading to the quest for understanding the origins and mechanisms of biological nitrogen fixation. Biological nitrogen fixation is a critical process that allows certain microorganisms, known as diazotrophs, to convert inert atmospheric N₂ into ammonia (NH₃), a biologically useful form of nitrogen. This conversion is essential for the growth and proliferation of various life forms. However, this biochemical feat is not without its challenges, primarily because the reaction requires energy and the catalytic action of metalloenzymes called nitrogenases. Traditionally, molybdenum (Mo) has been recognized as the key metal cofactor (FeMoco) for most nitrogenases. This raised an intriguing question: how did early life on Earth manage to perform biological nitrogen fixation when the bioavailability of molybdenum in the ancient oceans was limited to only a few nanomolar concentrations? This scarcity of soluble molybdenum appeared to contradict the prevailing notion that molybdenum-based nitrogenase (Mo-nitrogenase) evolved only when ample molybdenum became available. Recent evidence, however, suggested the presence of Mo-based nitrogenases in the mid-late Archean oceans long before the Great Oxidation Event, which occurred approximately 2.3-2.4 billion years ago. This discrepancy between the availability of soluble molybdenum and the existence of Mo-based nitrogenases created a paradox that demanded exploration.

Driven by the fundamental question of how life on Earth obtained the bioavailable nitrogen necessary for its existence, a new study published in the peer-reviewed Journal Environmental Science & Technology by Dr. Yizhi Sheng, Dr. Dongyi Guo, Dr. Shreya Srivastava, and led by Professor Hailiang Dong from the Department of Geology and Environmental Earth Science at Miami University in collaboration with Dr. Oliver Baars and Dr. Jason Whitham from North Carolina State University, the researchers conducted a series of experiments to investigate the role of mineral-bound trace metals in supporting anaerobic nitrogen fixation by microorganisms.

The researchers selected specific minerals and rocks that contained trace metals of interest, including Mo, vanadium (V), and iron (Fe). These minerals were chosen as potential sources of these essential metals. They also used the anaerobic diazotroph Clostridium kluyveri for their experiments. Diazotrophs are microorganisms capable of performing biological nitrogen fixation. They conducted incubation experiments in which they mixed the selected minerals and rocks with cultures of Clostridium kluyveri. These cultures were subjected to anaerobic conditions to mimic the environment in which anaerobic nitrogen fixation occurs. During the incubation experiments, the researchers monitored the release or mobilization of trace metals (Mo, V, and Fe) from the minerals into the surrounding culture medium. This step was essential to determine if the microorganisms could access these metals from the solid mineral sources.

The authors analyzed whether Clostridium kluyveri cells were able to take up the trace metals released from the minerals. They also investigated whether the microorganisms produced metal-chelating molecules known as siderophores or metallophores, which could facilitate the uptake of trace metals. To assess the impact of mineral-bound trace metals on nitrogen fixation, the researchers measured the expression of nitrogenase genes in Clostridium kluyveri. Nitrogenase is the enzyme responsible for converting atmospheric N₂ into NH₃ during nitrogen fixation. The team also  examined the physical interactions between the microbial cells and the mineral surfaces. This involved characterizing the cell-mineral associations and changes in mineral surface chemistry that might occur during the incubation experiments. Through these experiments, the researchers aimed to gain insights into whether mineral-bound trace metals, even under anaerobic conditions, could serve as a source of essential metals for biological nitrogen fixation. Their findings revealed the ability of microorganisms like Clostridium kluyveri to access and utilize these trace metals from solid mineral sources, challenging conventional notions of metal bioavailability and offering new perspectives on the mechanisms of anaerobic nitrogen fixation.

The results of the study have important implications for our understanding of nitrogen cycling on early Earth and the broader field of mineral-microbe interactions.   The authors unequivocally demonstrates that mineral-bound trace metals, including molybdenum, vanadium, and iron, can indeed serve as a source of these metals for anaerobic nitrogen fixation. This finding challenges the conventional belief that only soluble forms of trace metals are biologically accessible. It suggests that microorganisms have evolved mechanisms to extract metals from solid minerals, potentially reshaping our understanding of metal bioavailability in diverse environments. Moreover, the research highlights the resourcefulness of microorganisms in acquiring essential trace metals. Microbes like C. kluyveri employ various strategies, including siderophore production and direct contact with mineral surfaces, to obtain metals from solid sources. Understanding these microbial tactics could have broader implications for biogeochemical cycling in natural environments and potentially aid in addressing metal deficiencies in degraded ecosystems. The study also offers a potential solution to the paradox surrounding the presence of Mo-based nitrogenases in the ancient Archean oceans with low levels of soluble molybdenum. By demonstrating that microbial processes can access molybdenum from minerals, it suggests that biological nitrogen fixation may have occurred early in Earth’s history, even under conditions of limited soluble metal availability. The research raises questions about the role of mineral-bound trace metals in modern nitrogen cycling and its implications for ecosystems. Could microorganisms in contemporary environments be accessing trace metals from minerals in ways that we have not yet fully comprehended? This study beckons researchers to explore such possibilities in diverse ecosystems, from soils to deep-sea hydrothermal vents.

In summary, the study led by Professor Hailiang Dong and collaborators provides a captivating glimpse into the complex interaction between microorganisms and minerals, a partnership that has shaped the Earth’s biogeochemical cycles for eons. The revelation that mineral-bound trace metals can support anaerobic nitrogen fixation challenges our preconceived notions about metal bioavailability and opens up exciting avenues for future research. In the words of Professor Dong and Dr. Sheng, “Our findings underscore the incredible adaptability and resourcefulness of microorganisms in their quest for essential nutrients from solid minerals. They challenge us to explore the hidden mechanisms that sustain life on this planet and inspire us to continue unraveling the mysteries of our Earth’s past and present.”