Marine nitrogen fixation

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 (NH+4), nitrate (NO−3), and nitrite (NO−2).^[1] 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.^[2] 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.^[1]

History, context and research

Nitrogen was discovered in the 18th century, by the scientist Daniel Rutherford.^[3] Nitrogen's importance in agriculture, plant growth and the nitrogen cycle became clear. However, at this time, marine nitrogen fixation remained unexplored.^[3]

Biological nitrogen fixation was discovered by Herman Hellriegel and Hermann Wilfarth in 1880.^[3] They discovered that the root nodules of legumes host nitrogen-fixing bacteria which convert atmospheric nitrogen gas into forms the plants could use.^[3] 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.^[2] 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.^[4] The diversity and abundance of nitrogen-fixing bacteria, along with isotopic data and the distribution of specialized nitrogenase genes provide evidence supporting this theory.^[4]

Distribution of nitrogen fixation and balances

Divisions of the world's oceans centred on the South Pole. Animated to show the increased specificity of the division hierarchy.

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.^[5][6] 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.^[5]

In oligotrophic waters, organic nitrogen is highly limited, reaching concentrations as below 1 μmol/L in the open ocean of the Mediterranean Sea.^[7] 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.^[8][9] This species uses the enzyme nitrogenase to provide approximately 100 to 150 million tonnes of nitrogen per year globally in the open ocean.^[8][5][9]

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.^[5] 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.^[10] 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.^[10][11] Despite these processes, the Pacific is not devoid of fixation.^[5] 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.^[12][6]

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.^[5][12] Coastlines, which experience more upwelling in the higher latitudes, have less biotic nitrogen fixation.^[13] They instead rely on biologically available notrogen existing in the ecosystem available as DOM or within organisms susceptible to grazing.^[14] Additionally, Ammonium is made available in marine sediment via upwelling, with 76–83% organic nitrogen remineralized and redistributed by this process.^[15] Nitrogen concentrations, in the form of ammonia and nitrate, can exceed 100 μmol/L in coastal upwelling regions.^[13][5] 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.^[5] Nitrogen fixation rates in surface waters ranged from below detection limits to 7.51 nmol N L⁻¹ d⁻¹ in coastal water depending on these varied factors.^[16] Considering the seasonal variability of upwelling and nutrient abundance in costal waters means the fixation rates are dynamic with greater flux but on average, the productivity of costal ecosystems keeps nitrogen production low.^[13][14]

Sediment

Nitrogenase operates exclusively under anaerobic conditions,^[17] 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%.^[14] This is why a large amount of nitrogen fixation occurs within marine sediments. Fixation rates additionally vary with temperature of the sedimentary substrate.^[13] The concentration of O₂ also varies alongside nutrient availability, penetrating less as temperature increases increasing competition for resources amongst heterotrophs.^[14][6]

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.^[18] 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.^[19]

Nitrogen experiments use the quasi-conservative tracer N* (Nstar) to track the linear relationship between Nitrogen and Phosphorus nitrification and denitrification.^[19] 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.^[5][19]

Following the addition of N*, water samples are collected from various latitudes within the same gyre to track nitrogen distribution.^[5] 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.^[18] 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.^[5]

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,^[20] 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.^[16][12] 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.^[12][20]

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.^[12] 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.^[20]

Mesocosm and laboratory culture experiments

The variability of marine ecosystems forces the majority of nitrification and fixation research to be performed in the lab.^[19] Nitrification rates are calculated in a lab setting by comparing the concentrations of NO−2, NO−3, and NH+4 following incubation of filtered marine samples either in specific cultures or in bottled samples.^[8]

These experiments work for aquatic and sediment species dependent on their ability to survive in an enclosed system.^[14][6] Specific nitrogen-fixing species can be isolated on plates, but their often pseudo-anaerobic nature makes this difficult for many species.^[14] 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.^[21]

Processes in the ocean

Biochemical pathways

Marine nitrogen cycling pathways in different oceanic environments, specifically corals, open and deep ocean, oxygen minimum zones (OMZs), and sediments. Arrows indicate transformations of forms of nitrogen gas. Nitrogen fixation can be shown as the conversion of N₂ to NH+4. The black arrows represent nitrogen gas exchange with the atmosphere.

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 (PO3−4) and iron (Feⁿ⁺) to support their growth while converting atmospheric nitrogen gas (N₂) into ammonium (NH+4) and nitrate (NO−3), which are essential for many biological processes in the ocean.^[22] 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.^[23]

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.^[22]

Light

It serves as a vital energy source for photosynthetic nitrogen fixers, and insufficient intensity will reduce their ability to perform efficiently.^[22]

Oxygen levels

The nitrogenase enzymes encoded in nitrogen fixers are highly sensitive to oxygen.^[24] 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.^[25][26]

Temperature

When ocean temperatures rise, Trichodesmium populations tend to shift toward higher latitudes, potentially leading to a decline in their presence in tropical regions.^[23] These changes could significantly affect nitrogen availability, global distribution of nitrogen-fixing species,^[22] and disrupt overall ecosystem productivity across different oceanic regions.^[23]

Seasonality

Seasonality plays a large role in the variability of all other factors. Fixation is lowest in the summer, particularly in coastal regions,^[5][14] due to the influence temperate summer conditions have on the process of upwelling.^[13][15] Upwelling is strongest in the summer months: April to August in the northern hemisphere and December to February in the southern hemisphere.^[27] This flood of available nitrogen is complemented by blooms of photosynthetic species, so that cellular production is increased overall and DON (dissolved organic nitrogen) is greatly increased, reducing the need for fixation.^[5]

Free-living marine nitrogen fixation species

There is a diverse range of marine species that contribute as nitrogen fixers to ensure a continuous supply of bioavailable nitrogen for primary production.

Filamentous cyanobacteria

Trichodesmium is particularly important in nutrient-poor (sub)tropical surface waters; and, unlike many others, it does not form heterocysts. Instead, it uses alternative mechanisms to regulate oxygen levels during nitrogen fixation.^[28]

Unicellular cyanobacteria

UCYN-A is unique in that it lacks photosystem II, which allows it to fix nitrogen during the day while avoiding oxygen interference from photosynthesis.^[28] In contrast, Crocosphaera watsonii has adapted to perform nitrogen fixation at night when oxygen levels are low, reducing the risk of nitrogenase inhibition.^[29]

Heterocyst-forming species

Organisms including Nodularia spp. and Anabaena spp. have developed heterocysts, which are thick-walled, structurally distinct cells that create an anaerobic environment necessary for nitrogenase enzyme in nitrogen fixers.^[25][30]

Symbiotic diazotrophs

They contribute significantly to nitrogen fixation in oligotrophic regions.^[22] For example, Richelia intracellularis is a heterocystous cyanobacterium that forms symbiotic relationships with diatom Hemiaulus hauckii. Diazotroph resides within the diatom's frustule, where it benefits from stable conditions and access to nutrients. In return, the diatom host receives a steady supply of nitrogen and therefore continues to thrive in nitrogen-depleted waters.^[26] This partnership enhances nitrogen availability in marine ecosystems, promotes more diatom growth, and enables the biogeochemical cycling on a global scale.

Examples of major nitrogen-fixing symbionts

Organisms form various symbiotic relationships, including mutualism, commensalism, and parasitism.^[31][32] Many symbiotic diazotrophs exhibit mutualism with the host cell, the benefit being receiving protein source and sufficient nutrients in return for supplying a biologically available form of nitrogen to the host.^[33]

Cyanobacterial symbionts

Cyanobacteria, the oldest known photosynthetic prokaryotes, form symbiotic relationships with various unicellular and multicellular organisms which exhibit diverse metabolic pathways, including diatoms, dinoflagellates and haptophytes.^[34][35] Unicellular and filamentous cyanobacteria are the main forms of cyanobacteria observed to have symbiotic relationships.^[35]

Candidatus Atelocyanobacterium thalassa (UCYN-A)

Scanning electron micrograph of Braarudosphaera bigelowii. Haptophyte algae, coccolithophore that forms symbiotic relationships with Candidatus Atelocyanobacterium thalassa. Found in open ocean and coastal environments.

Candidatus Atelocyanobacterium thalassa, otherwise called UCYN-A, is found to form close symbiosis with haptophyte algae, Braarudosphaera bigelowii, and their relationship is described as obligate endosymbiosis.^{[28][25][36][37]} Various nutrients, such as amino acids, purines, vitamins, and carbon sources in the form of glucose and glycerol-3-phosphate transferred from the host to UCYN-A, which is essential for its metabolic function and nitrogen assimilation.^[28] In return, UCYN-A provides fixed nitrogen as ammonium,^[25] ammonia, alanine, and glycine.^[28] Additionally, reduced genome size, loss of genes for carbon uptake and photosynthesis, increase in gene expression for nitrogen fixation for UCYN-A,^[36][33] as well as a decrease in ammonium uptake by B. bigelowii^[25] further represents the adaptation of both species to depend on this symbiotic relationship.

Richelia

The image of Hemiaulus—R. intracellularis symbiosis obtained by environmental high content fluorescence microscopy (eHCFM).

Richelia intracellularis is a filamentous cyanobacterial diazotroph^[38][39] that has endosymbiotic relationships with diatoms, described as diatom–dinotroph associations (DDAs).^[40][39][41] Rhizosolenia and Hemiaulus^[38][39] are diatom species that have a close association with R. intracellularis.^[40][41] The domination of Hemiaulus-R. intracellularis symbiosis is seen in various pelagic systems, marking a greater contribution to the nitrogen and carbon fixation.^[38] Evidence of obligate relationships could be seen from a positive correlation between nitrogen assimilation by R. intracellularis and photosynthesis by the host diatom,^[38][39] and successful growth of the host diatom without the addition of nitrogen in a laboratory setting.^[39]

Calothrix spp.

Filamentous heterocystous cyanobacterium, Calothrix spp. is also part of the DDAs, which form an ectosymbiotic relationship with diatoms, mainly Chaetoceros, attached to their spines.^[42][43] Neutral metabolism dependency was suggested through increasing nitrogen fixation rate compared to its free form^[43] and growth restriction.^[42]

Non-cyanobacterial symbionts

Candidatus Tectiglobus diatomicola is classified under Rhizobia, a heterotrophic nitrogen-fixing proteobacterium symbiont that has an obligate endosymbiotic relationship with a pennate diatom Haslea.^[44] Rhizobia has been known to have facultative endosymbiotic relationship with terrestrial legume.^[45] The reduced genome size and low transcription of glycolysis-related genes of Ca. T. diatomicola suggest that it relies on Haslea on bypassing glycolysis. Haslea also relies on ammonium sources, as 99% of fixed nitrogen is transferred to Haslea.^[44]

Evolution to the organelle

The evolutionary development of symbiotic relationships is found in many cellular mechanisms, such as chloroplasts in photosynthetic eukaryotes.^[35] The gradual evolution of organelles from endosymbiotic relationships does not have a distinguishing point that indicates the difference.^[46] Researchers use various criteria as indicators of organelle transformation, including genetic integration, cellular integration, and metabolic integration.^[46] However, categorization becomes especially challenging when curtain endosymbiosis is in the process of transformation, as indicators of both endosymbiosis and organelle may coexist.^[46][47]

Diazoplast

Due to the insufficient genetic integration and little to no endosymbiotic gene transfer (EGT) to the host diatom, diazoplasts could be classified as endosymbiotic symbionts.^[48][49] However, completion of high metabolic and cellular integration may be the indicator of organelle. Diazoplasts are found within the cells of the diatom Epithemia and are present in all species of this genus.^[48] This constant existence of intercellular diazotroph, high degree of nutrient dependence on host,^[48] and absence of regulation for nitroganase synthesis^[49] may indicate the organelle-like function.

Nitroplast

While some metabolic co-dependence between UCYN-A and Braarudosphaera bigelowii can be the evidence for both symbiosis and organelle, the level of genetic integration, generally assessed through the amount of genes transferred to the organelle and those lost from it,^[46] is suggested by the significant portion of UCYN-A proteins that are encoded from B. bigelowii.^[33] The cellular integration, including the close association of UCYN-A and other organelles in the B. bigelowii during cell division^[33] and consistent size correlation of UCYN-A with B. bigelowii, also suggests the organelle function.^[50]

Industrial and human implications

Anthropogenic nitrogen in oceans

Since 1850, the rate of anthropogenic reactive nitrogen deposition has doubled in oceanic systems,^[51] driven by human activities such as agriculture, fossil fuel combustion, aquaculture, and household and industrial waste disposal.^[52] Agriculture in the United States alone contributes about 11 million tonnes of nitrogen through the use of fertilisers.^[53] Through agricultural runoff, this nitrogen seeps through soil into waterways and eventually reaches marine ecosystems.^[54] Similarly, atmospheric nitrogen from fossil fuel combustion is deposited into water systems through precipitation or dry deposition, before being carried into the ocean by streams.^[55] In aquaculture, dissolved nitrogen waste primarily comes from unused feed and fish waste.^[56] Sewage, of which about 80% is left untreated, is sometimes disposed of directly into oceans.^[57]

Consequences of excess nitrogen

Algae bloom in Lake Chapala, México

Excess nitrogen concentrations in marine ecosystems can lead to toxic algae blooms, a loss of biodiversity, marine dead zones and shellfish poisoning.^[16] Toxic algae blooms occur when excess nitrogen and phosphorus allow algae and phytoplankton to grow uncontrollably on surface water. They rapidly consume oxygen and block sunlight from penetrating deeper water in a process called eutrophication.^[58] As a result, the local biodiversity is greatly reduced, especially in bottom-dwelling species. This may lead to the formation of "dead zones", areas which have a dissolved oxygen concentration of less than 2 mL of O₂/litre and thus cannot support marine life. Geological evidence suggests most dead zones today did not exist prior to anthropogenic nitrogen deposition, but rather studies show that their numbers have increased exponentially (doubled each decade) since the 1960s.^[59] The Gulf of Mexico Dead Zone, in the northern Gulf of Mexico, is one of the world's largest hypoxic areas,^[60] with excess nitrogen flowing in from the mouth of the Mississippi River. Although the Gulf of Mexico Dead Zone is considered seasonal, records indicate that since 1985 it continues to occur increasingly frequently and in larger areas, negatively impacting marine life and the many dependent fisheries.^[61]

Surviving shellfish can become contaminated. As filter-feeders, they filter and absorb toxins produced by the phytoplankton associated with algal blooms. These toxins are difficult to eliminate, as they are not destroyed by cooking or freezing, and thus can cause severe disease in humans when ingested.^[62]

Nitrogen pollution also affects tourism in areas dependent on boating and fishing activities. Each year the United States tourism industry loses an estimated US$1 billion due to nitrogen-induced algal blooms.^[63]

Mitigation strategies

Mitigation strategies to reduce marine anthropogenic nitrogen deposition vary depending on the source of pollution. To reduce agricultural runoff, practices such as the use of winter cover crops and perennial cropping systems have successfully mitigated nitrogen leakage. Most agricultural runoff occurs during winter and spring, when moisture levels drop and evapotranspiration rates decrease.^[64] Winter cover crops protect soil during these seasons, and perennial crops such as alfalfa, allow stronger nitrogen retention. Growing these on agricultural land has reportedly resulted in 3 times and 30–50 times less nitrogen leaching, respectively.^[33] Policy and educational measures to reduce excessive and unnecessary fertilisation have also been implemented.^[33]

To reduce nitrogen pollution from human and industrial activities, the more widespread use of advanced denitrification wastewater plants is suggested. Currently, the cost of building and maintaining such facilities prevents the majority of wastewater produced globally from being adequately treated.^[65] More efficient nitrogen removal and recovery processes are currently being developed to address this issue.^[66]

Other mitigation strategies that have been implemented include the construction of artificial habitats and waterway rehabilitation.^[33] Habitats such as wetlands and lagoons act as nitrogen sinks and prevent its constant seepage into the ocean. Similarly, waterways such as rivers and lakes can be rehabilitated to improve their nitrogen retention. They then act as buffers, reducing marine nitrogen fluxes.^[33]