NITROUS OXIDE

2.3.1 Properties and roles in agriculture

Nitrous oxide is a colorless water-soluble and non-toxic gas. It is commonly known as laughing gas due to its euphoric effects while inhaling. It is a significant dissociative anesthetic in surgery and dentistry (Thomson et al., 2012). Apart from this, N2O is a detrimental greenhouse gas and ozone depleting gas. 90% of the atmospheric N2O is removed by photolysis in stratosphere (see equation (1)) (Reay et al., 2007). While the remaining N2O undergoes oxidation reactions with oxygen (O) producing NO which will further participate in the ozone depleting reaction (see equation (2) and (3)) (Ravishankara, Daniel and Portmann, 2009). Both sink pathways are slow and lead to its long atmospheric lifetime of 121 years (IPCC, 2014b). Therefore, N2O is 298 times stronger in Global Warming Potential (GWP) than that of CO2 in 100-year time horizon (IPCC, 2013). Ozone depleting induces a series of health problems such as sunburn, genetic mutation, cataracts and other health problems and N2O is likely to play the dominant role in ozone depletion in future (Portmann, Daniel and Ravishankara, 2012).

𝑁₂𝑂 →

ℎ𝑣𝑁₂+ 𝑂 (1) 𝑂 + 𝑁₂𝑂 → 𝑁𝑂 + 𝑂₂ (2) 𝑁𝑂 + 𝑂₃→ 𝑂₂+ 𝑁𝑂₂ (3)

𝑁𝑒𝑡: 𝑂 + 𝑂₃→ 2𝑂₂ (4)

Since Haber–Bosch process was available to artificially fix nitrogen as ammonia in 1908, intensive farming based on synthetic fertilizer has been thriving and N2O emissions drastically increased due to the enrichment of reactive nitrogen in soils (Thomson et al., 2012). Nitrogen is an essential element for all forms of life to biosynthesize molecules such as amino acids, proteins, nucleic acids and other substances. All the nitrogen circulated in biosphere ends up in the atmosphere as dinitrogen (N2) that is the most abundant and stable atmospheric molecule with a percentage of 78.08 (Thomson et al., 2012). The triple bonds in dinitrogen need 10-3 KJ M-1 to break thus effective catalysts are needed to accelerate the molecule division (Thomson et al., 2012). All accessible redox states are involved in nitrogen cycles entailing -3 (NH3), -2(NO), -1(NH2OH), 0(N2), +1(N2O), +3(NO2-) to +5(NO3-) (Thomson et al., 2012).

N2O is biologically generated from specific classes of bacteria, archaea and fungi residing both in soil and ocean as a metabolite of respiration and energy producing (Thomson et al., 2012).

Processes included are nitrification, denitrification, nitrate ammonification (dissimilatory nitrate reduction to ammonia, DNRA), anaerobic ammonia oxidation (anammox) and other pathways (Shoun and Tanimoto, 1991; Wrage et al., 2001; Prendergast-Miller, Baggs and Johnson, 2011;

Marcel, Marchant and Kartal, 2018). Bacteria denitrification and ammonia oxidation from both agricultural and natural soils contribute to roughly 62% of N2O emissions around 6 and 4.2 Tg N year-1, respectively, the other one-third of the emissions is from the ocean dominated by archaea (Thomson et al., 2012). Besides. a tiny amount of N2O is produced from non-biological process such as chemical decomposition of NO2- (chemidenitrification) and hydroxylamine (NH2OH) oxidation (Bremner, Blackmer and Waring, 1980; Bremner, 1997).

Agricultural soil plays the largest anthropogenic N2O source (Del Grosso, 2010). 80% of atmospheric N2O increase attributes to food production (IPCC, 2013). The usage of nitrogen fertilizer in agriculture is still increasing (WMO, 2019). The world population increased by nearly 90% from 3.6 to 6.9 billion from 1970 to 2010 and productivity increased 230% due to additive usage of nitrogen fertilizer from 32 Mt to 106 Mt (IPCC, 2014a). Consequently, N2O emission accounted for 38% of the agricultural GHG emissions in 2010, which had increased by 73% since 1970 (IPCC, 2014a). The atmospheric N2O concentration was increasing averagely at a rate of 0.93 ppb per year, reaching 329.9 ± 0.1 ppb by 2017 (WMO, 2019).

In agriculture, the most significant biological N2O emissions are nitrification and denitrification from soils (Thomson et al., 2012). Ammonia oxidizing bacteria and nitrite oxidizing bacteria dominates autotrophic aerobic nitrification while denitrifying bacteria mediate anaerobic denitrification (Hayatsu, Tago and Saito, 2008). Other microbes that involve in nitrification and denitrification are anammox bacteria that convert NH4+ and NO2- to N2 under anaerobic conditions.

Some fungi also process denitrification and co-denitrification that produces N2O and N2 (Hayatsu, Tago and Saito, 2008). Archaea also promote soil denitrification (Santoro et al., 2011).

2.3.2 Nitrification and denitrification

Atmospheric nitrogen (N2) is fixed as ammonia (NH3) naturally only by free-living and symbiotic bacteria and archaea (diazotrophs). The triple bond is divided under the catalyst of nitrogenase generating ammonia. Nitrogenases is the only family that known catalyze this paramount step in nitrogen fixation. There are three variants of nitrogenase enzymes, each of which contains a metal ion of molybdenum, iron or vanadium and they possess complex, unique iron and Sulphur clusters (Thomson et al., 2012).

The ammonium ion (NH4+) is oxidized to nitrate (NO3-) in nitrification with three steps. Firstly, ammonium is oxidized to hydroxylamine (NH2OH) catalyzed by ammonia monooxygenase (AMO), during which N2O is produced. AMO is a transmembrane copper protein. It is an endergonic reaction under the presence of O2 (see equation (5)). The second step is hydroxylamine oxidized to nitrite (NO2-). It had been believed that this step was catalyzed by octaheme hydroxylamine oxidoreductase (HAO). However, it was found that HAO produces nitric oxide (NO) instead of nitrite. Nitrite is derived from nitric oxide by further unknown process (see equation (6)) (Caranto and Lancaster, 2017). The last step is nitrite oxidized to nitrate under the catalyst of nitrite oxidoreductase (NXR) that is a membrane-associated iron-sulfur molybdoprotein (see equation (7)) (Meincke et al., 1992; Spieck et al., 1998). Both ammonia oxidizing bacteria (AOB) and ammonia oxidizing archaea (AOA) participate in the ammonia oxidation in soils while AOA dominate N2O production in marine environment (Leininger et al., 2006; Wuchter et al., 2006; Hatzenpichler, 2012).

𝑁𝐻₃ + 𝑂₂ + 2𝐻⁺+ 2𝑒⁻ →

𝐴𝑀𝑂𝑁𝐻₂𝑂𝐻 + 𝐻₂𝑂 (5) 𝑁𝐻₂𝑂𝐻 + 𝐻₂𝑂 →

𝐻𝐴𝑂+𝑢𝑛𝑘𝑛𝑜𝑤𝑛𝑁𝑂₂⁻+ 5𝐻⁺+ 4𝑒⁻ (6)

𝑁𝑂₂⁻+ 𝐻₂𝑂 →

𝑁𝑋𝑅𝑁𝑂₃⁻+ 2𝐻⁺+ 2𝑒⁻ (7)

Nitrate and nitrite ion generated from nitrification is readily reduced by denitrification which is a stepwise reduction from nitrate to N2 with the presence of four enzymes (Zumft, 1997; Bothe, Ferguson and Newton, 2006). The first step is the reduction from nitrate to nitrite catalyzed by nitrate reductase (Nar) (see equation (8)). The second step is nitrite reduced to nitric oxide catalyzed by nitrite reductase (Nir) (see equation (9)). nitric oxide is further reduced to N2O catalyzed by Nitric oxide reductase (Nor) (see equation (10)). N2O is converted into N2 as a termination of denitrification catalyzed by Nitrous oxide reductase (Nos) (see equation (11)). Nitrous oxide reductase is the only known enzyme that governs the reaction from N2O to N2 (Zhang et al., 2019).

Thus, failure of the enzyme leads to the termination as N2O rather than N2. This is a crucial implication in respect of N2O emissions in agriculture concerning N fertilizer application.

𝑁𝑂₃⁻+ 2𝐻⁺+ 2𝑒⁻ →

𝑁𝑎𝑟𝑁𝑂₂⁻+ 𝐻₂𝑂 (8) 2𝑁𝑂₂⁻+ 4𝐻⁺+ 2𝑒⁻ →

𝑁𝑖𝑟 2𝑁𝑂 + 2𝐻₂𝑂 (9) 2𝑁𝑂 + 2𝐻⁺+ 2𝑒⁻ →

𝑁𝑜𝑟𝑁₂𝑂 + 𝐻₂𝑂 (10) 𝑁₂𝑂 + 2𝐻⁺+ 2𝑒⁻ →

𝑁𝑜𝑠𝑁₂+ 𝐻₂𝑂 (11)

The majority of denitrifying bacteria is anoxic or facultative aerobic heterotrophs. nitrate acts as an electron acceptor when oxygen is limited (Zumft, 1997). Both nitrification and denitrification are pathways for denitrification bacteria to generate ATP (Bothe, Ferguson and Newton, 2006).

2.3.3 Influential factors to nitrification and denitrification

Both processes of nitrification and denitrification are governed by complex conditions respecting microbes, soil properties and plant species. The related factors include e.g., N availability, aeration, temperature, moisture, organic carbon content, C:N ratio, texture, pH, soil management, metal cofactors (Signor and Cerri, 2013), soil type (Stevens and Laughlin, 1998), earthworm activities (Karsten and Drake, 1997; Borken, Brumme and Xu, 2000; Speratti, Whalen and Rochette, 2007) and other factors.

Fertilization is a crucial part of field management that influence both nitrification and denitrification thus further impact N2O emissions. N Fertilizer modifies soil N content by

increasing available ammonia (NH4+) and nitrate (NO3-). It is reported that the higher amount of NH4+-N, the greater nitrification process (Mosier, 2001; Khalil, Mary and Renault, 2004; Liu et al., 2005) Thus, more N2O is converted from NH4+ through various intermediate reactions of nitrification. Secondly, when nitrate amounts and other conditions are favorable, denitrification releases N2O subsequently to nitrification (Carmo et al., 2005; Ruser et al., 2006; Zanatta et al., 2010). In addition, lack of oxygen during ammonia oxidation would encourage NO2- as an electron acceptor instead of O2 to produce N2O or NO (Signor and Cerri, 2013). Last, the promoted biomass production by fertilization increases leftovers on field, which facilitates N2O emission in a long term (Hellebrand, Scholz and Kern, 2008). N addition might lead to soil organic C depleting because of the promotion of both soil mineralization and microbial activities (Signor and Cerri, 2013). As soil organic carbon is a basic feedstock for microbial growth and activities, soil organic C positively correlated with N2O emissions (Brentrup et al., 2000), the higher available soil organic carbon, the higher N2O emissions when N and moisture are not limiting factors (Ruser et al., 2006).

The N2O emission is mostly from the rhizosphere because root leachate promotes heterotrophic activities and denitrifiers compete with ammonifiers for this carbon. As a result, N2O emissions are influenced by both the organic carbon availability and the competence of the two microbial groups (Kim et al., 2004; Morley and Baggs, 2010).

There are other field management concerning fertilization that would affect N2O emissions such as N in-depth application and splitting application. There is not uniformed result about the influence of those applications since the studies are highly case specialized including specific crop types, meteorological conditions and other detailed environmental conditions. But in general, the lower available oxygen and nitrogen, the more N2O ends up as N2 (Brentrup et al., 2000; Yang and Cai, 2007). The longer N2O remains in soil, the more chance it is reduced to N2 as an electron acceptor regardless of soil properties (Chapuis‐Lardy et al., 2007). Therefore, in-depth application prevents N2O emission than surface application. Both in-depth and splitting application avoid nitrogen surface runoffs by precipitation and irrigation, which prevent N2O emissions (Signor and Cerri, 2013).

Cutting N addition to deduce the N2O emission is not feasible since the insufficient N supplement leads to SOC decomposition and poor productivity (Jaynes and Karlen, 2005). It is found that N2O emissions from agriculture could be reduced with no or little yield penalty by reducing N fertilizer inputs to levels that just satisfy crop needs (McSwiney and Robertson, 2005). Enhanced and

sustained soil productivity optimize crop production per hectare and reduce overall GHG emissions by avoiding land use change from other natural lands, such as wetlands, peatlands and forests, into farmlands (Jaynes and Karlen, 2005).

Temperature and moisture are crucial factors for both nitrification and denitrification since they govern microbial activities. They also influence N2O diffusion from soil to the atmosphere (Davidson and Swank, 1986). A close relation between N2O emission and air as well as soil temperature was reported (Wolf and Brumme, 2002; Zhang and Han, 2008). Increasing soil temperature will stimulate soil respiration thus creating anaerobic environment for denitrification (Signor and Cerri, 2013). Soil N2O emissions exponentially increase in response to the increase of soil temperatures (0-50ºC) (Liu et al., 2011). Soil moisture also intervenes in both nitrification and denitrification (Davidson and Swank, 1986). The extremely high moisture prohibits N2O emissions due to the inhibition of microbial activity while alternating dry and moist period might promote N2O emissions (Brentrup et al., 2000). When soil moisture is high, fewer soil pores are filled with air so that N2O emitted from denitrification is thriving (Brentrup et al., 2000). It also complies to non-fertilized field as non-fertilized field has a baseline N2O emission derived from mineralization of SOM (Del Grosso et al., 2006). Thus, rain and irrigation event might increase N2O emissions.

Perdomo observed N2O emission promotion after rain during high soil temperature period and Liu found the N2O diffusion clearly increased after rain and returned back to normal after three days (Liu et al., 2006; Perdomo, Irisarri and Ernst, 2009).