Agritech to Tame the Nitrogen Cycle

Abstract

While the Haber–Bosch process for N-fixation has enabled a steady food supply for half of humanity, substantial use of synthetic fertilizers has caused a radical unevenness in the global N-cycle. The resulting increases in nitrate production and greenhouse gas (GHG) emissions have contributed to eutrophication of both ground and surface waters, the growth of oxygen minimum zones in coastal regions, ozone depletion, and rising global temperatures. As stated by the Food and Agriculture Organization of the United Nations, agriculture releases ∼9.3 Gt CO₂ equivalents per year, of which methane (CH₄) and nitrous oxide (N₂O) account for 5.3 Gt CO₂ equivalents. N-pollution and slowing the runaway N-cycle requires a combined effort to replace chemical fertilizers with biological alternatives, which after a 10-yr span of usage could eliminate a minimum of 30% of ag-related GHG emissions (∼1.59 Gt), protect waterways from nitrate pollution, and protect soils from further deterioration. Agritech solutions include bringing biological fertilizers and biological nitrification inhibitors to the marketplace to reduce the microbial conversion of fertilizer nitrogen into GHGs and other toxic intermediates. Worldwide adoption of these plant-derived molecules will substantially elevate nitrogen use efficiency by crops while blocking the dominant source of N₂O to the atmosphere and simultaneously protecting the biological CH₄ sink. Additional agritech solutions to curtail N-pollution, soil erosion, and deterioration of freshwater supplies include soil-free aquaponics systems that utilize improved microbial inocula to enhance nitrogen use efficiency without GHG production. With adequate and timely investment and scale-up, microbe-based agritech solutions emphasizing N-cycling processes can dramatically reduce GHG emissions on short time lines.

CURRENT STATE OF THE GLOBAL N-CYCLE

Humanity is quickly approaching the moment where decisions on how we manage land use, distribution of planetary resources, and food supplies will determine our fate in the face of rapid climate change. It is clear that global inequality, wherein the wealthiest 10% of individuals are responsible for as much usage of resources and ecological damage as the bottom 80%, must be addressed if we are to avoid (or minimize) the more dire consequences to the human population and habitability of the planet (Rammelt et al. 2023). Without adjustments to our global food systems, negative environmental effects are expected to increase by 50%–90% from 2010 to 2050 due to further additions to the human population and a concomitant rise in global income (Springmann et al. 2018). The greatest environmental effect will be increased greenhouse gas (GHG) emissions followed by demands for arable land, water usage, and application of nitrogen and phosphorous fertilizers. Planetary boundaries that have already been crossed due to our current food systems include climate change, biosphere integrity, and N-cycle and phosphorous cycle thresholds (Rockstrom et al. 2009; Springmann et al. 2018). Additional boundaries that will be crossed soon include freshwater use, ocean acidification, land-use change, and destabilization of ecosystem functions that support human life.

Global food production is at the heart of our survival, which relies on delivering nutrients (e.g., nitrogen and phosphorous) to crops across diverse geographical regions. Prior to industrialized society, nitrogen delivery to plants was accomplished by microbial N₂-fixation and other natural processes. However, our relationship to the N-cycle changed when we discovered how to chemically fix atmospheric N₂ into reactive-N via the Haber–Bosch process (Galloway et al. 2014). Between 1850 and 1950, human-derived reactive-N (HdRn) was proportional to the population. From 1950 to 1980, the Green Revolution resulted in a marked uptick in HdRn, from about 12 to 30 kg N yr⁻¹ capita⁻¹. This was also a period of rapid population growth, peaking at around a 2% growth rate in the 1960s (Ritchie et al. 2023). From 1980 to the present day, HdRn has stabilized to around 30 kg N yr⁻¹ capita⁻¹, but due to a population of 8 billion and rising, this level of HdRN exceeds the planetary boundaries of sustainability, causing increased ecosystem degradation and unmitigated GHG production (Rockstrom et al. 2009; Galloway et al. 2014).

Some practical solutions to slow the crossing of planetary sustainability boundaries include shifting food choices toward plant-based diets, reducing food waste, and improving technology and management of food systems (Springmann et al. 2018). In addition, there is increasing interest in agritech solutions aimed toward direct reduction of GHG emissions while alleviating other environmental stresses (e.g., water and land demands). For instance, seaweed-based feed ingredients are under development to control CH₄ emissions from enteric fermentation in food animals (Vijn et al. 2020). Several companies are marketing microbial solutions to replace synthetic nitrogen and phosphorus fertilizers. Scaled-up soil-free systems that minimize water, chemical, land, and energy usage are emerging in urban centers. However, globalizing agritech requires overcoming substantial hurdles, including global social inequality, diversity of regional needs, geographical variations in ecosystems, and availability of investment and scale-up opportunities.

There are three main thresholds that must be considered for taming the global N-cycle: N-deposition rate, N-concentration in surface waters, and N-concentration in groundwaters (Schulte-Uebbing et al. 2022). However, extensive regional variation for these thresholds complicates mitigation strategies as Europe, China, India, and the United States experience N-excess, while N-deficiency is widespread in sub-Saharan Africa, Central and South America, and Southeast Asia. In N-deficient locations, none of the N-cycle thresholds have yet been crossed (Schulte-Uebbing et al. 2022). Biologically, there are also vast differences in how soils, plants, and microorganisms interact to manage nitrogen use efficiency depending on the health status of the soil. For instance, in nutrient-poor soils, plants have a strong dependence on fertilizer-N, whereas richer soils have ecologically complex networks of microbiota, plants, and minerals that support elaborate cycling and uptake of organic-N (Grandy et al. 2022). Thus, re-engineering of agroecosystems depends on regional differences in soil nutrition, management, and climate that can be partially addressed using a systems-biology understanding of plant-microbe-mineral interactions that impact regional nutrient cycles (Grandy et al. 2022).

MICROBIOLOGY OF THE N-CYCLE

Over the past several decades, researchers have made great strides in understanding the mechanisms, pathways, and ecology of the microbial N-cycle and the processes that generate N₂O (Stein 2019, 2020; Prosser et al. 2020). Chemolithotrophic ammonia oxidizers and denitrifiers are predominant and direct producers of N₂O in agroecosystems, although microbes encoding “clade II nitrous oxide reductase (NosZ)” enzymes can act as N₂O sinks as these microbes can use N₂O as their sole terminal electron acceptor (Fig. 1). Synthetic fertilizer usage has the strongest positive effect on the activity of ammonia-oxidizing bacteria (AOB), as these microorganisms tend to prefer inorganic over organic ammonia as their sole energy source (Hink et al. 2018a,b). Furthermore, AOB, unlike their archaeal counterparts (AOA), can reduce nitrite to N₂O enzymatically and are thus relatively efficient at producing N₂O as a terminal product; ca. up to 10% of ammonia-N can be transformed to N₂O-N by AOB given ample substrate and with low oxygen conditions (Stein 2019).

Figure 1.

N-cycle processes and feedbacks in soil that contribute to N₂O. Biological nitrification inhibitors (BNIs) block the activity of AMO or HAO enzyme complexes (and can also block methane monooxygenases). Nitrification is the main nitrous oxide (N₂O)-producing process under oxic conditions. Nitrifier denitrification, performed by ammonia-oxidizing bacteria (AOB), produces N₂O under hypoxic conditions. Respiratory denitrification is the main N₂O-producing process under anoxic conditions and is driven by microbes that consume organic carbon. The process of “clade II N₂O reduction” removes N₂O to N₂ under anoxic conditions by microorganisms that consume organic carbon. (AMO) Ammonia monooxygenase, (HAO) hydroxylamine dehydrogenase, (NOO) nitric oxide oxidase, (NIR) nitrite reductase, (NXR) nitrite oxidoreductase, (NOR) nitric oxide reductase, (NAR) nitrate reductase, (NosZ) nitrous oxide reductase.

The product of ammonia oxidation by AOB and AOA is nitrite, which is further oxidized to nitrate by nitrite-oxidizing bacteria to complete the process of nitrification. Comammox bacteria oxidize ammonia to nitrate in one step, earning their title as “complete ammonia oxidizers” (Stein 2019). Nitrate is a primary pollutant and marker of N-saturation, contributing to eutrophication and contamination of surface and groundwaters that leads to oxygen minimum zone formation in coastal regions. Nitrate is also the first terminal electron acceptor for denitrification, which is the other major microbial source of N₂O (Fig. 1). As nitrification is both a direct and indirect source of N₂O, and a competitor for fixed nitrogen by plants, inhibitors of the nitrification process are essential for regions suffering from N-saturation effects (Norton and Ouyang 2019). While several options for nitrification inhibition have been explored, the use of biological nitrification inhibitors (BNIs) seems to hold promise for mitigating N₂O emissions and causing the least harm to soil health (Nardi et al. 2020).

AGRITECH TO MITIGATE N₂O FROM AGRICULTURE

Biofertilizer is a rapidly growing, microbe-based, agritech venture, with profitable companies emerging worldwide. Biofertilizers make use of microorganisms that are natural members of the plant microbiome to augment seed germination and growth, resistance to pathogens, and overall yields and crop quality (Satish Kumar et al. 2022). The success of biofertilizers relies on the systematic evaluation of plant–microbe relationships and the composition of the rhizosphere microbiome. Beneficial microorganisms targeted for biofertilizers possess traits including the ability to degrade organic matter, enhancement of nutrient accessibility, such as phosphorous and nitrogen, production of phytohormones, and increased resistance to pathogen infection along with protection from other stresses. Once beneficial microorganisms are identified, they can be further engineered or used as natural products to apply directly to crops.

Some commercialized classes of biofertilizers include microbes that increase nitrogen fixation as free-living, associative, or symbiotic partners; microbes that solubilize phosphate, zinc, or potassium; sulfur-oxidizing microbes; and rhizobacteria that promote plant growth through secreted growth factors (Satish Kumar et al. 2022). Successful application of biofertilizer microbes into soils requires close attention to their quality, longevity, activity, purity, and associated carrier material to ensure their survival and adequate stimulation of crop growth and improved health (Satish Kumar et al. 2022). Although biofertilizers have been successfully produced and deployed as a general soil amendment for a variety of crop and soil types, the diversity of plant–microbe relationships across agroecosystems may need to be reconfigured from one farm to the next. In other words, a biofertilizer that works well for corn farmers in the United States might not work for rice farmers in the Philippines, thus requiring further research into the microbiome that naturally associates with target crops across diverse geographic regions.

A related crop amendment to biofertilizers is a group of compounds known as BNIs. Nitrification is the primary competitor with crops for fertilizer nitrogen, which has led to the application of synthetic nitrification inhibitors (SNIs), mainly nitrapyrin, dicyandiamide (DCD), and 3,4-dimethylpyrazole phosphate (DMPP) (Subbarao and Searchinger 2021). While these chemicals are effective in increasing nitrogen use efficiency and reducing N₂O emissions, they are also unstable and mainly affect the activity of AOB, and not AOA, which are often more dominant and active than AOB in soils (Leininger et al. 2006). Furthermore, SNIs can have deleterious effects on nontarget microbes in soils (Nardi et al. 2020), causing unintended alterations to soil functions. Contrary to SNIs, BNIs are naturally produced molecules exuded by plant roots into the rhizosphere to enable them to compete with nitrifiers for available ammonium (Nardi et al. 2020). BNIs target a variety of enzymes and structures in the microbial cell including the enzymes of nitrification (ammonia monooxygenase and hydroxylamine dehydrogenase), membranes, central metabolites (e.g., nitric oxide), and quorum-sensing molecules (Nardi et al. 2020).

Agritech ventures aim to identify BNIs produced naturally by target crops that inhibit both AOB and AOA. For application, BNI production in situ by crops can be enhanced using genetic engineering tools, or by direct amendment of soils with industrially produced BNIs. Key research required to bring BNIs to the marketplace includes (1) ensuring their efficacy, either alone or in cocktails, in both improving nitrogen use efficiency and in preventing N₂O production, (2) ensuring that they do not impair other soil services by affecting nontarget microbiota, and (3) ensuring that they do not target methane oxidizers that act as the sole biological CH₄ sink (Stein and Klotz 2011). To this last point, BNIs may dramatically reduce N₂O emissions while stimulating CH₄ emissions due to inhibition of the homologous enzyme complexes, ammonia and methane monooxygenase (Klotz and Norton 1998).

Soil-free cropping systems are another agritech solution that addresses multiple sustainability boundaries, including nitrogen and phosphorous cycles and freshwater use, among others. The most promising emergent soil-free technology is aquaponics, which integrates fish with crop production (Goddek et al. 2019). In recirculating aquaponics systems, fish waste is converted by microorganisms to vital plant nutrients, mainly nitrate, and the plants regenerate clean water for the fish. These enclosed systems can operate at low energy, low water usage, and occupy minimal land space while producing substantial amounts of leafy green crops and fish protein (Goddek et al. 2019). Although aquaponics technology is immature, it is clear that further developments to improve nitrogen use efficiency and microbiome function to avoid N₂O production and pathogen infection is needed, along with consideration of socioeconomic aspects for integrating soil-free farming in the context of regional and geographic diversity (Junge et al. 2017). There is also a need to better explore plant-microbe interactions and improvements to microbial inocula to expand the variety of crops that can be produced at high efficiency while improving nitrogen use efficiency and reducing N₂O emissions.

SYNERGIES BETWEEN N-CYCLE AGRITECH WITH OTHER APPLICATIONS

Many other agritech solutions, including crop engineering for enhanced photosynthesis, synthetic biology solutions to improve rates of carbon fixation, reduction of stomata in food crops, and increasing carbon-efficient tree plantations, are vital to slow the crossing of planetary sustainability boundaries in food production systems. However, keen attention to the microbial N-cycle in all agritech has the capacity to mitigate N₂O emissions and improve overall nitrogen use efficiency by crops, thus bringing the N-cycle back toward sustainability. The N-cycle is intimately connected to the carbon cycle, such that impacts or interference with one cycle will necessarily impact the other. For instance, the microbial processes responsible for N₂O and CH₄ emissions are both sensitive to redox and nutrient balance and are also heavily influenced by the availability of trace metals like copper and iron (Stein 2020). As an example, methanotrophic bacteria produce the copper-chelating chalkophore, methanobactin, that prevents access to copper for the expression of nitrous oxide reductase enzymes (NosZ) by denitrifiers (Chang et al. 2021). Thus, when copper is at low availability, CH₄ oxidation will proceed at the expense of N₂O reduction, leading to higher N₂O emissions with lower CH₄ emissions. Similarly, some methane oxidizers use nitrate as a terminal electron acceptor, leading to N₂O as a final product (Kits et al. 2015); the result is a reduction in CH₄ emission with a proportional increase in N₂O emission when nitrate is readily available and low redox conditions promote its usage. Therefore, technologies aimed at reducing CH₄ emissions must consider how nutrient amendments, like copper and nitrate, effect microbial processes leading to N₂O emissions, and vice versa.

Microbial agritech ventures are undergoing a revolution that aims to bring the N-cycle back toward sustainability. The success of the technologies discussed above rely heavily on investment and scale-up, specific regional and geographic needs, and attention to global socioeconomic inequalities. Research focused on systems biology, the integration of plant-microbe-mineral interactions, and microbiome function is necessary to achieve widespread development, optimization, and deployment of microbial agritech. Specific attention to the microbial N-cycle and its interrelationships to other nutrient cycles, including carbon, is essential for disrupting our current food production systems and replacing them with highly efficient, biology-based systems that work within the boundaries of planetary sustainability.