Nitrogen fixation is a vital biochemical process that supports the productivity of marine environments. It involves the conversion of nitrogen gas (N2) to forms available to living organisms such as ammonia (NH3), ammonium (), nitrate (), and nitrite (). Since nitrogen is a limiting nutrient in most marine ecosystems, nitrogen fixation plays a key role in sustaining primary production, particularly in oligotrophic regions. Currently about 13 prokaryote genera are known to fix nitrogen. Understanding marine nitrogen fixation is crucial to the study of global nitrogen cycling. Research indicates an imbalance between nitrogen fixation and denitrification rates, impacting nitrogen availability in different oceanic regions.
History, context and research Nitrogen was discovered in the 18th century, by the scientist Daniel Rutherford. Nitrogen's importance in agriculture, plant growth and the nitrogen cycle became clear. However, at this time, marine nitrogen fixation remained unexplored.
Biological nitrogen fixation was discovered by Hermann Hellriegel and Hermann Wilfarth in 1880. They discovered that the root nodules of legumes host nitrogen-fixing bacteria which convert atmospheric nitrogen gas into forms the plants could use. In the 20th century, tracing techniques including nitrogen-15 isotopes were used to study nitrogen fixation in aquatic environments. Specifically, cyanobacteria such as Trichodesmium were found to be major nitrogen fixers in the ocean. This confirmed microbes' role as nitrogen fixers in aquatic environments.
While less common than bacteria, nitrogen fixation has also been identified within some Archaea among methanogenic species. Historically, there has been a debate regarding how Archaea acquired the ability to fix nitrogen. One hypothesis, the bacteria-first hypothesis, suggests that the nitrogen fixation process evolved in bacteria before being transferred to Archaea via horizontal gene transfer. The diversity and abundance of nitrogen-fixing bacteria, along with isotopic data and the distribution of specialized nitrogenase genes provide evidence supporting this theory.
Distribution of nitrogen fixation and balances
Open ocean Nitrogen fixers are widespread throughout the world's oceans, but vary in density depending on the abundance of nutrients, light availability, oxygen levels, temperature and season. The highest density of marine nitrogen fixers is observed in the pelagic zone, where oligotrophic conditions require the fixation of inorganic dinitrogen (N2) via biological processes.
In oligotrophic waters, organic nitrogen is highly limited, reaching concentrations as below 1 μmol/L in the open ocean of the Mediterranean Sea. Fixation is the only way to convert atmospheric nitrogen to an organic form usable by the surrounding ecosystem. The deep waters of the subtropical and tropical latitudes of the North Atlantic ocean, as well as the Mediterranean Sea, have the highest levels of fixation, dominated by the filamentous cyanobacteria Trichodesmium spp. This species uses the enzyme nitrogenase to provide approximately 100 to 150 million tonnes of nitrogen per year globally in the open ocean.
The Pacific Ocean, especially at higher latitudes, acts as a sink for organic nitrogen. This is primarily due to the direct correlation between O2 levels and nitrogen fixation rates. The Pacific ocean performs Pacific meridional overturning circulation (PMOC) which pulls warmer waters from the Atlantic and subtopics into the upper latitudes of the Pacific. By the time it reaches the deep ocean however, it is depleted of oxygen and filled with organic waste which creates large oxygen minimum zones (OMZ) where fixation is low and denitrification is high. Despite these processes, the Pacific is not devoid of fixation. This ocean is dominated by single cell diazotrophs, particularly UCYN-A, which are capable of nitrification and can reach concentrations as high as 106 cells per litre, even in oligotrophic waters.
Coastal Fixation rates are lower in coastal systems compared to those of open waters. This is the result of a variety of factors, foremost being nutrient concentrations. Coastlines, which experience more upwelling in the higher latitudes, have less biotic nitrogen fixation. They instead rely on biologically available nitrogen existing in the ecosystem available as DOM or within organisms susceptible to grazing. Additionally, ammonium is made available in marine sediment via upwelling, with 76–83% organic nitrogen remineralized and redistributed by this process. Nitrogen concentrations, in the form of ammonia and nitrate, can exceed 100 μmol/L in coastal upwelling regions. The increased concentrations of available nitrogen mean that even species capable of fixation will rely on existing materials, suspending the process of the more costly fixation, thereby reducing rates. The variation is also dependent on the concentrations of other macronutrients, with particular dependence on the N:P ratio with species needing both for proper biosynthesis. Nitrogen fixation rates in surface waters ranged from below detection limits to in coastal water depending on these varied factors. Considering the seasonal variability of upwelling and nutrient abundance in coastal waters means the fixation rates are dynamic with greater flux but on average, the productivity of coastal ecosystems keeps nitrogen production low.
Sediment Nitrogenase operates exclusively under anaerobic conditions, and while most species have systems to allow the mitigation of this problem, many nitrogen fixers have their highest production within anaerobic conditions. Additionally, marine sediments display lower concentrations of both nitrogen and phosphorus, particularly at lower depths. At only 6 cm depth, available nitrogen decreases by as much as 50%. This is why a large amount of nitrogen fixation occurs within marine sediments. Fixation rates additionally vary with temperature of the sedimentary substrate. The concentration of O2 also varies alongside nutrient availability, penetrating less as temperature increases increasing competition for resources amongst heterotrophs.
Research methodologies Stable isotope tracer experiments Stable isotope tracers allow for both macro and micro scaled experiments. Using a variety of different isotopes, researchers can observe the distribution and uptake of different nutrients in diverse ecosystems. Traditionally, these experiments track the incorporation of these radio nutrients into specific organisms but with the aid of satellite imaging and large scale sampling, they can track the incorporation into biomass of an ecosystem as a whole.
Nitrogen experiments use the quasi-conservative tracer N* (Nstar) to track the linear relationship between Nitrogen and Phosphorus nitrification and denitrification. N* is a stable Nitrogen tracer that displays a linear association with marine phosphorus and therefore provides a means of visualizing nitrogen and fixation in marine systems on a large scale.
Following the addition of N*, water samples are collected from various latitudes within the same gyre to track nitrogen distribution. This method allows for the visualization of nitrogen distribution on a large scale and is often supplemented with satellite imaging to gather accurate distribution data. Additionally, these experiment are used to calculate global fixation rates as well as the flux within these rates making it one of the best large scale research methods.
Quantitative PCR (qPCR) and RT-PCR PCR has a long standing relationship in genetic and microbiological experiments. By identifying genetic markers with functions specific to nitrogen fixation processes allows researchers to express and quantify those genes within marine samples, allowing for approximate measurements of marine fixers density in varied ecosystems.
By quantifying the planktonic nifH genes through amplification, nitrogen fixation can be estimated in both filtered samples and laboratory cultures tracking the change over time under varied conditions. This process can also be used to identify and quantify species that cannot be cultured as the genetic markers are organism specific and are present in sample even following the lysis of cells.
Despite being easier, specifically with modern equipment, this method only provides estimates, not accurate counts due to bottling and other manipulatory effects present in most laboratory experiments. Additionally, it only shows the density of microbes with the potential to perform nitrogen fixation, not the actual rate of expression. While this technique is a powerful tool, it is often supplemented with other tools for accurate measurements and analysis.
Mesocosm and laboratory culture experiments The variability of marine ecosystems forces the majority of nitrification and fixation research to be performed in the lab. Nitrification rates are calculated in a lab setting by comparing the concentrations of , , and following incubation of filtered marine samples either in specific cultures or in bottled samples.
These experiments work for aquatic and sediment species dependent on their ability to survive in an enclosed system. Specific nitrogen-fixing species can be isolated on plates, but their often pseudo-anaerobic nature makes this difficult for many species. Mesocosm experiments are not performed in the presence of diverse marine systems. Instead, their controlled, laboratory based methodology means that some factors of the traditionally dynamic marine systems may be missed and therefore, an understanding of the whole picture remains elusive.
Processes in the ocean
Biochemical pathways
Nitrogen fixation is a complex biochemical process that requires energy and is influenced by various environmental factors. In the surface ocean, nitrogen fixers consume phosphate () and iron (Fen+) to support their growth while converting atmospheric nitrogen gas (N2) into ammonium () and nitrate (), which are essential for many biological processes in the ocean. As remineralization occurs, these microorganisms release phosphate, iron, and nitrate into the water column and their carbon-rich biomass sinks to deeper waters, contributing to the oceanic carbon cycle.
Environmental factors influencing marine nitrogen fixation
Nutrient availability A crucial factor for nitrogen fixers. Maintaining adequate levels of iron and phosphorus has been shown to be critical for microorganisms to carry out nitrogen fixation effectively.
Light It serves as a vital energy source for photosynthetic nitrogen fixers, and insufficient intensity will reduce their ability to perform efficiently.
Oxygen levels The nitrogenase enzymes encoded in nitrogen fixers are highly sensitive to oxygen. To overcome this challenge, some microorganisms have evolved adaptations to form specialized cells called heterocyst (create a low-oxygen environment) that allow nitrogen fixation to occur spatially different from the oxygen-producing photosynthesis processes.
Temperature When ocean temperatures rise, Trichodesmium populations tend to shift toward higher latitudes, potentially leading to a decline in their presence in tropical regions. These changes could significantly affect nitrogen availability, global distribution of nitrogen-fixing species, and disrupt overall ecosystem productivity across different oceanic regions.
Seasonality