Microbial nitrogen transformation processes across environments: more than just a cycle

Nitrogen (N) is unique amongst the bioessential elements in that its major reservoir is in the atmosphere, as triple-bonded N2, which comprises 78% of the atmosphere. Breaking the extremely strong triple bond of N2 is challenging; in nature, only the microbial nitrogenase enzyme is known to be capable of this reaction, and nitrogenase is by far the dominant source of N2 fixation on the modern Earth. Abiotic N2 fixation is found only in extremely high energy situations, namely lightning bolts, and industrial N2 fixation for fertilizer production. Indeed, N is one of the elemental cycles most influenced by human activities; in a little over a century, humans have more than doubled fixed N2 in the environment due to industrial N2 fixation via the Haber–Bosch process. Excess fertilizer use and subsequent runoff contributes to eutrophication of waterways and emissions of greenhouse gases, namely nitrous oxide. While humans have radically changed the N cycle in the Anthropocene, it was single-celled microorganisms that established the nitrogen cycle and its enzymatic processes over Earth's history [1.Stüeken E.E. et al.The evolution of Earth's biogeochemical nitrogen cycle.Earth Sci. Rev. 2016; 160: 220-239Crossref Scopus (0) Google Scholar]. Once fixed into 'reactive' N (the term for all forms of N in the environment except N2), N cycles through dozens of inorganic and organic nitrogenous forms on the Earth's surface [2.Zhang X. et al.Global nitrogen cycle: critical enzymes, organisms, and processes for nitrogen budgets and dynamics.Chem. Rev. 2020; 120: 5308-5351Crossref PubMed Scopus (167) Google Scholar]. With every passing year, it becomes clearer that the so-called N cycle, often drawn as a circle, is instead a tangled web. While relatively stable chemical species like nitrate and ammonium are often the focus of attention, highly reactive intermediates – such as nitric oxide, hydroxylamine, and even hydrazine (rocket fuel) – are increasingly recognized as the linchpins in the N web but often experience cryptic cycling due to their reactivity. Even more complexity is introduced when one considers the fact that the N web is woven by biotic–abiotic couplings, such as chemodenitrification, in which products of microbial metabolism react with ferrous iron to produce gaseous N. Biotic transformations in the N cycle are primarily catalyzed by metalloenzymes, many of which contain iron, molybdenum, and/or copper cofactors [3.Lycus P. et al.Structural biology of proteins involved in nitrogen cycling.Curr. Opin. Chem. Biol. 2023; 74102278Crossref Scopus (0) Google Scholar]. For instance, alternative forms of nitrogenase (vanadium–iron or iron–iron) have been detected, but they are rare in natural environments. Intriguingly, molybdenum–iron nitrogenase is by far the dominant form, even in environments in which vanadium is present at higher concentrations than molybdenum [4.Warren M.J. et al.Molybdenum-based diazotrophy in a sphagnum peatland in northern Minnesota.Appl. Environ. Microbiol. 2017; 83e01174–17Crossref Scopus (43) Google Scholar]. In some environments, such as the low-chlorophyll, high-nitrate regions of the oceans, scarcity of trace metals, particularly iron, limits photosynthesis and nitrate assimilation by phytoplankton. Scientific disciplines separate the N cycle/web into soil, freshwater, marine, and anthropogenic compartments; microbial life, of course, ignores all such designations and N freely flows across such artificial boundaries. While challenging, enhanced communication across disciplinary spheres is vital for expediting scientific discoveries and applications. Towards that end, Trends in Microbiology has joined forces with iScience to present a special issue on 'Nitrogen Cycling Across Environments' that explores microbial N processes across aquatic and terrestrial environments, with a focus on lessons that can be gained from cross-communication across the fields. The N cycle is replete with examples of how processes discovered in one environment have informed another. This is particularly common in the study of wastewater engineering, in which a large priority is conversion of reactive N from human and animal waste to N gas while minimizing greenhouse gas emissions, also known as biological N removal [5.Winkler M.K. Straka L. New directions in biological nitrogen removal and recovery from wastewater.Curr. Opin. Biotechnol. 2019; 57: 50-55Crossref PubMed Scopus (184) Google Scholar]. Anaerobic oxidation of ammonium (anammox) was first characterized in samples of wastewater sludge but has since been discovered in soil as well. More recently, the pathway of complete nitrification, thus far restricted to terrestrial environments, was discovered in other samples from engineered, aquatic systems. Crosstalk between fields can also bring new ideas about potential interactions between species in the N cycle. Interactions between diazotrophic bacteria and photosynthetic eukaryotes in root nodules (e.g., in legumes and alder trees) have long been studied in soils but remained relatively unknown in the ocean until the discovery of UCYN-A unicellular cyanobacteria in symbiosis with haptophyte algae. Likewise, while non-cyanobacterial diazotrophs have long been known to be key for terrestrial N2 fixation, they were only relatively recently discovered in the ocean. The role of viruses in the nitrogen cycle remains understudied across ecosystems. Many technological challenges hinder progress, such as challenges cultivating and genetic systems for environmentally key species and applying limited rate measurements across scales. Methods for microbial cultivation and pathway characterization are increasingly complex, which poses problems for widespread access to study of the cycle. This special issue tackles these questions and more. Three of the articles focus on the entry of N into the cycle via N2 fixation catalyzed by nitrogenase. Chakraborty et al. discuss engineering N2-fixing bacteria to increase N yield to crops, Zehr and Capone highlight recent developments and challenges in marine N2 fixation research, and Rucker and Kaçar describe interconnections between metal cycles and N cycles. Bowen et al. highlight salt marshes as an interface between terrestrial and marine realms, both subject to anthropogenic disturbances, which remove excess N prior to its entry into marine habitats. Deutsch and colleagues tackle the temperature dependence of biological N2 fixation and uncover a relationship that is similar across terrestrial and marine environments. Beeckman et al. critically examine the role of nitrification inhibitors that are used to relieve the deleterious effects associated with excess nitrification, providing insights into their applicability in the field. Saghaï and Hallin review ammonifiers in terrestrial and aquatic environments. The article looks at enzymes involved in the reduction of nitrate and nitrite to ammonia with a particular focus on the widely used marker, NrfA. The special issue also highlights two recently published articles on the influence of tillage practices on the rhizosphere [6.Behr J.H. et al.Long-term conservation tillage with reduced nitrogen fertilization intensity can improve winter wheat health via positive plant–microorganism feedback in the rhizosphere.FEMS Microb. Ecol. 2024; 100fiae003Crossref Scopus (1) Google Scholar] and the identification of Methylomirabilis bacterium capable of methane-dependent complete denitrification [7.Yao X. et al.Methane-dependent complete denitrification by a single Methylomirabilis bacterium.Nat. Microbiol. 2024; 9: 464-476Crossref PubMed Scopus (5) Google Scholar]. Finally, our Microbe of the Month for this special issue is the diazotrophic Gram-negative bacterium, Bradyrhizobium japonicum (diazoefficiens), covered by Ong and O'Brian. We would also like to draw your attention to the articles published in iScience that are part of this interdisciplinary collection (https://www.cell.com/cp/collections-microbial-nitrogen-transformation). Christensen and Rousk review global sources of N2O emissions, identifying those which are influenced by anthropogenic activities, and highlighting agricultural systems with the potential for mitigation. Derikvand and colleagues focus on the microbial ecology of engineered aquaponic systems, investigating microbial community dynamics and ammonia removal in response to ammonia-oxidizing inocula and changing pH. While dozens of microbial reactions in the N cycle have been discovered, many more exergonic and disproportionation reactions, and the enzymes that catalyze them, await unearthing [8.Kuypers M.M.M. et al.The microbial nitrogen-cycling network.Nat. Rev. Microbiol. 2018; 16: 263-276Crossref PubMed Scopus (2225) Google Scholar]. Crystal structures of membrane proteins such as ammonia monooxygenase have been notoriously difficult to solve but are essential for mechanistic understanding of nitrogen cycle processes [9.Bradely B.T. et al.Integrated structural biology and molecular ecology of N-cycling enzymes from ammonia-oxidizing archaea.Environ. Microbiol. 2017; 5: 484-491Google Scholar]. Forms of oxygen-resistant enzymes previously thought to be extremely oxygen sensitive have also been discovered, such as the atypical form of nitrous oxide reductase, and surely many more remain to be found [10.Simon J. Klotz M.G. Diversity and evolution of bioenergetic systems involved in microbial nitrogen compound transformations.BBA-Bioenergetics. 2013; 2: 114-135Crossref Scopus (259) Google Scholar]. As we venture even further into the Anthropocene, the importance of studying N cycling across environments subject to global climate change becomes ever more important. Nitrogen is a bioessential element with an almost limitless supply from the atmosphere, yet conversion to reactive N currently relies on fossil fuels and results in eutrophication and greenhouse emissions. Harnessing insights from microbes that drive the N cycle to tackle these environmental challenges will be increasingly key going forward [11.Stein L.Y. Klotz M.G. The nitrogen cycle.Curr. Biol. 2016; 26: R94-R98Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar].