Di­a­zo­ma­nia III – Ni­trous Ox­ide, Please Don't Laugh About It

This is the third and fi­nal in­stall­ment of this year's Di­a­zo­ma­nia. The prior two Mon­days I dis­cussed the nitro­gen cy­cle and the ni­tro­gen fixer Tri­chodesmium.

by Roberto

Fig­ure 1. Empty gas can­is­ter on a bike la­ne. Source

If you bike a lot, par­tic­u­larly if you race, you learn to al­ways carry along a spare in­ner tube and a CO₂ can­is­ter to help you rapidly change a flat. So, when I started notic­ing empty gas can­is­ters while rid­ing along a few of the ubiq­ui­tous Co­penhagen bike lanes, the first thing that came to mind was: "watch out, peo­ple must get a lot of flat tires around here." But it made no sense; there seemed to be too many canis­ters and most Danes are not prone to leav­ing trash around. Then I no­ticed that I en­coun­tered these for­lorn ob­jects more fre­quently along the lanes tra­vers­ing the "seed­ier" parts of town (if you can call them that in a city as im­pec­ca­ble as Copen­hagen). I looked into the mat­ter, even­tu­ally re­al­iz­ing that the can­is­ters had not con­tained CO₂, but rather N₂O, ni­trous ox­ide. It was news to me that ni­trous ox­ide recre­ational use was back en vogue, a large scale reprise of Humphry Davy's "laugh­ing gas par­ties" at the turn of the 18th cen­tury. Nowa­days, os­ten­si­bly sold as an agent for mak­ing whipped cream and other culi­nary foams, many of these can­is­ters end up de­livering a laugh­ing party drug. De­spite its use­ful­ness in den­tistry as an anx­i­olytic, I have to say that ni­trous ox­ide is "no laugh­ing mat­ter." Be­cause ni­trous ox­ide is a pow­er­ful green­house gas.
 

Fig­ure 2. Sim­pli­fied global ni­trous ox­ide bud­get. Source

Ni­trous ox­ide is the third most abun­dant green­house gas in the at­mos­phere af­ter car­bon di­­oxide and methane. While the amount of ni­trous ox­ide is nowhere near that of car­bon di­oxide, it has a warm­ing po­ten­tial nearly 300-fold greater, mak­ing its green­house ef­fect quite large in­deed. Where does all that ni­trous ox­ide come from? From a global per­spective, about half of it comes from an­thro­pogenic sourc­es. Mind you, that is not be­cause hu­mans syn­the­size huge amounts of it. It is be­cause cer­tain hu­man prac­tices lead to its in­creased pro­duc­tion. As the pop­u­la­tion in­creased, large scale agri­cul­tural prac­tices and – to a lesser ex­tent – waste­water treat­ment also in­creased, and ni­trous ox­ide emis­sions in­evitably in­creased. Re­call that since the in­ven­tion of the Haber-Bosch process to con­vert ni­tro­gen gas into am­mo­nia a lit­tle over one hun­dred years ago, the use of syn­thetic fer­til­izer explod­ed. As we (over)use an es­ti­mated 100 Tg of fer­til­izer in in­dus­trial agri­cul­ture a year, soil and wa­ter mi­crobes ready to use that fixed ni­tro­gen will do just that. Therein lies the prob­lem.

Fig­ure 3. Den­i­tri­fi­ca­tion gen­er­ally pro­ceeds through some com­bi­na­tion of the fol­low­ing half re­ac­tions, with the en­zyme cat­alyz­ing the re­ac­tion in paren­the­ses. Source

Ni­trous ox­ide forms part of the ni­tro­gen cy­cle. Den­i­tri­fy­ing mi­crobes con­vert ni­trate to ni­tro­gen gas. This den­i­tri­fi­ca­tion does not hap­pen in a sin­gle step. Rather, the con­ver­sion is through a com­bi­na­tion of half re­ac­tions cat­alyzed by dif­fer­ent re­duc­tases. Ni­trate is first re­duced to ni­trite. Ni­trite can then be re­duced to ni­tric ox­ide (NO). Two mole­cules of NO are trans­formed into ni­trous ox­ide which in turn is re­duced to the fi­nal prod­uct, ni­tro­gen gas. But, NO and N₂O be­ing gases, some frac­tion of those pro­duced dur­ing den­i­tri­fi­ca­tion es­capes into the at­mos­phere. Yet, den­i­tri­fy­ing bac­te­ria do not ap­pear to be the ma­jor pro­duc­ers of ni­trous ox­ide. That role seems to be played – some­what sur­pris­ingly to me at least – by the ni­tri­fiers.

Fig­ure 4. Path­ways lead­ing to NO and N₂O pro­duc­tion in bac­te­r­ial ni­tri­fiers. Abi­otic N₂O pro­duc­tion in blue ar­rows. En­zy­matic N₂O pro­duc­tion in red ar­rows: CytL, cy­tochrome P460, NOR, ni­tric ox­ide re­duc­tase, NIR, ni­trite re­duc­tase. Black ar­rows, steps in am­mo­nia ox­i­da­tion to ni­trite: AMO, am­mo­nia monooxy­ge­nase; HAO, hy­drox­y­lamine ox­i­dore­duc­tase. Ad­apt­ed from Source

Ni­tri­fiers carry out the two main ox­i­da­tion steps that con­vert am­mo­nia to ni­trite (NO₂) and ni­trite to ni­trate (NO₃) (for an over­view see Di­a­zo­ma­nia I). They are thus the key mi­crobes that en­counter and deal with the mas­sive in­crease in am­mo­nia in soils due to cur­rent agri­cul­tural prac­tices. This has led Michael Wag­ner, a lead­ing fig­ure in ni­tri­fier re­search, to re­fer to them as "the gate­keep­ers of the ni­tro­gen cy­cle." And what these bac­te­ria (and ar­chaea) do with all that am­mo­nia is fas­ci­nat­ing. The ox­i­da­tion of am­mo­nia to ni­trite in­volves three steps (shown in black in the fig­ure). The two in­ter­me­di­ates, hydroxyl­ami­ne (NH₂OH) and ni­tric ox­ide (NO) can serve as sub­strates to pro­duce ni­trous ox­ide (N₂O). Through abi­otic mech­a­nisms, a low amount of ni­trous ox­ide is al­ways pro­duced. But, most of the ni­trous ox­ide is pro­duced when ni­tri­fi­ca­tion is ac­tu­ally run­ning in re­verse, in a process para­dox­i­cally named "ni­tri­fier denitri­fication," where ni­trite is re­duced to ni­trous ox­ide via ni­tric ox­ide by the cor­re­spond­ing re­duc­tases NIR and NOR. In ad­di­tion, a re­cent pa­per showed that the long-known cy­tochrome P460 (CytL) of the ni­tri­fier Ni­tro­somonas eu­ro­pea cat­alyzes the con­ver­sion of both hydroxyl­amine and ni­tric ox­ide into ni­trous ox­ide. The pres­ence of these ni­trous ox­ide-form­ing path­ways in nearly all am­mo­nia ox­i­diz­ing bac­te­ria sug­gested that for­ma­tion of this green­house gas will al­ways be as­so­ci­ated with ni­tri­fi­ca­tion.

That no­tion changed with the iso­la­tion of bac­te­ria that carry out both steps of ni­tri­fi­ca­tion, ammo­nia ox­i­da­tion and ni­trite ox­i­da­tion. As I men­tioned be­fore, for more than a cen­tury mi­cro­bi­ol­o­gists were un­der the im­pres­sion that these two steps had to be car­ried out by sep­a­rate mi­crobes. Then came the dis­cov­ery and cul­tur­ing of Ni­tro­spira in­opinata that sin­gle­hand­edly car­ries out the com­plete ox­i­da­tion of am­mo­nia to ni­trate (COMAMMOX). Hav­ing a pure cul­ture of the or­gan­ism, re­search­ers set out to de­ter­mine just how much ni­trous ox­ide it pro­duced. An ex­cit­ing re­cent pa­per sug­gests that COMAMMOX are in­deed "green" ni­tri­fiers in that they pro­duce very lit­tle ni­trous ox­ide, only through the abi­otic path­way. A key in­sight came from com­par­ing the genome of N. in­opinata to sev­eral genomes from can­di­date (un­cul­tured) COMAMMOX as well as genomes from con­ven­tional am­mo­nia ox­i­diz­ers. The genes en­cod­ing ni­tric ox­ide re­duc­tase (NOR) and cyto­chrome P460 (CytL) were al­most uni­ver­sally miss­ing from COMAMMOX, strongly sug­gest­ing that these bac­te­ria will not make ni­trous ox­ide. They fol­lowed these com­par­a­tive ge­nomics stud­ies with ex­ten­sive phys­i­o­log­i­cal ex­per­i­ments with N. in­opinata demon­strat­ing its in­abil­ity to pro­duce ni­trous ox­ide en­zy­mat­i­cally. Will this dis­cov­ery prove help­ful in re­duc­ing ni­trous ox­ide emis­sions due to agri­cul­ture? The au­thors point to­wards a pos­si­ble so­lu­tion: "Con­se­quently, adop­tion of con­di­tions that fa­vor the growth of com­plete ni­tri­fiers over am­mo­nia ox­i­diz­ing bac­te­ria, for ex­ample, in en­gi­neered sys­tems and in soils where co­mam­mox bac­te­ria have been iden­ti­fied, may in­flu­ence ni­tri­fi­ca­tion-de­pen­dent N₂O emis­sions." That cer­tainly sounds like a fea­si­ble ap­proach. In my view, how­ever, the root of the prob­lem still lies in the overuse and abuse of syn­thetic fer­tilizers world­wide. Sadly, es­ti­mates are that less than 20% of the ni­tro­gen ap­plied as fer­til­izer ends up in the food we eat. The rest mostly leaches or goes up into the air. Es­tab­lish­ing more ef­fi­cient ways of us­ing syn­thetic fer­til­iz­ers in agri­cul­ture would go a long way to­wards al­le­vi­at­ing the ni­trous ox­ide prob­lem. If that were to hap­pen, I would not laugh, but I would cer­tainly smile.