Break­ing ni­tro­gen while gen­er­at­ing meth­ane

In­sights into a “hot” mi­crobe that can grow on ni­tro­gen while pro­du­cing meth­ane

24-Nov-2022 - Germany

Scientists at the Max Planck Institute for Marine Microbiology have successfully enhanced cultivation of a microorganism that can fix nitrogen (N2) while producing methane (CH4) and ammonia (NH3) and investigated exciting details of its metabolism.

© Max Planck Institute for Marine Microbiology

Nevena Maslać with batch cultures of Methanothermococcus thermolithotrophicus in the Microbial Metabolism lab, which allow for precise testing of different growth conditions in complete absence of oxygen.

Car­bon and ni­tro­gen are es­sen­tial ele­ments of life. Some or­gan­isms take up key po­s­i­tions for the cyc­ling of both of them – among them Methanothermococcus thermolithotrophicus. Be­hind the com­plic­ated name hides a com­plic­ated mi­crobe. M. thermolithotrophicus is a mar­ine heat-lov­ing meth­ano­gen. It lives in ocean sed­i­ments, from sandy coasts and salty marshes to the deep-sea, prefer­ably at tem­per­at­ures around 65 °C. It is able to turn ni­tro­gen (N2) and car­bon di­ox­ide (CO2) into am­mo­nia (NH3) and meth­ane (CH4) by us­ing hy­dro­gen (H2). Both products, am­mo­nia and meth­ane, are very in­ter­est­ing for bi­o­tech­no­lo­gical ap­plic­a­tions in fer­til­izer and bio­fuels pro­duc­tion.

Tristan Wag­ner and Nevena Maslać from the Max Planck In­sti­tute for Mar­ine Mi­cro­bi­o­logy have now man­aged to grow this mi­crobe in a fer­menter – a chal­len­ging en­deav­our. “It is very com­plic­ated to provide the per­fect con­di­tions for this mi­crobe to thrive while fix­ing N2 – high tem­per­at­ures, no oxy­gen and keep­ing an eye on hy­dro­gen and car­bon di­ox­ide levels”, says Maslać, who car­ried out the re­search as part of her PhD pro­ject. “But with some in­genu­ity and per­sever­ance, we man­aged to make them thrive in our lab and reach the highest cell dens­it­ies re­por­ted so far.” Once the cul­tures were up and run­ning, the sci­ent­ists were able to in­vest­ig­ate the physiology of the mi­crobe in de­tail, and later on deepen their study by look­ing how the meta­bol­ism from the mi­crobe ad­apts to the N2-fix­a­tion. “In close col­lab­or­a­tion with our col­leagues Chandni Sidhu and Hanno Teel­ing, we com­bined physiolo­gical tests and dif­fer­en­tial tran­scrip­tom­ics, which al­lowed us to dig deeper into the meta­bol­ism of M. thermolithotrophicus”, Maslać ex­plains.

As im­prob­able as a bumble­bee

The meta­bolic abil­it­ies of M. thermolithotrophicus are puzz­ling: These mi­crobes use meth­ano­gen­esis, a meta­bol­ism that ori­gin­ated on the early an­oxic Earth, to ac­quire their cel­lu­lar en­ergy. Com­pared to hu­mans that use oxy­gen to trans­form gluc­ose into car­bon di­ox­ide, meth­ano­gens ob­tain only a very lim­ited amount of en­ergy from meth­ano­gen­esis. Para­dox­ic­ally, fix­ing ni­tro­gen re­quires gi­gantic amounts of en­ergy, which would ex­haust them. “They are a bit like bumble­bees, which are the­or­et­ic­ally too heavy to fly but ob­vi­ously do so, nev­er­the­less”, says senior au­thor Tristan Wag­ner, group leader of the Max Planck Re­search Group Mi­cro­bial Meta­bol­ism. “Des­pite such en­ergy lim­it­a­tion, these fas­cin­at­ing mi­crobes have even been found to be the prime ni­tro­gen fix­ers in some en­vir­on­ments.”

A ro­bust ni­tro­genase

The en­zyme that or­gan­isms use to fix ni­tro­gen is called ni­tro­genase. Most com­mon ni­tro­genases re­quire Mo­lyb­denum to per­form the re­ac­tion. Mo­lyb­denum ni­tro­genase is well-stud­ied in bac­teria liv­ing as sym­bionts in plant roots. Their ni­tro­genase can be in­hib­ited by tung­state. Sur­pris­ingly, the Bre­men sci­ent­ists found that M. ther­mo­li­tho­trophi­cus is not dis­turbed by tung­state while grow­ing on N2. “Our mi­crobe was only de­pend­ent on mo­lyb­denum to fix N2 and not bothered by tung­state, which im­plies an ad­apt­a­tion of metal-ac­quis­i­tion sys­tems, mak­ing it even more ro­bust for dif­fer­ent po­ten­tial ap­plic­a­tions”, says Maslać.

Re­think­ing am­mo­nia pro­duc­tion

Ni­tro­gen fix­a­tion, i.e., gain­ing ni­tro­gen from N2, is the ma­jor pro­cess to in­sert ni­tro­gen into the bio­lo­gical cycle. For in­dus­trial fer­til­izer pro­duc­tion this pro­cess is car­ried out via the Haber-Bosch pro­cess, which ar­ti­fi­cially fixes ni­tro­gen to pro­duce am­mo­nia with hy­dro­gen un­der high tem­per­at­ures and pres­sures. It is used to pro­duce most of the world’s am­mo­nia, an es­sen­tial fer­til­izer to sus­tain global ag­ri­cul­ture. The Haber-Bosch pro­cess is ex­tremely en­ergy-de­mand­ing: It con­sumes 2% of the world’s en­ergy out­put, and re­leas­ing at the same time up to 1.4% of global car­bon emis­sions. Thus, people are look­ing for more sus­tain­able al­tern­at­ives to pro­duce am­mo­nia. “The pro­cess used by M. thermolithotrophicus shows that out there in the mi­cro­bial world there are still solu­tions that might al­low for a more ef­fi­cient pro­duc­tion of am­mo­nia, and that they can even be com­bined with bio­fuel pro­duc­tion through meth­ane”, says Wag­ner. “With this study, we un­der­stood that un­der N2-fix­ing con­di­tions, the meth­ano­gen sac­ri­fices its pro­duc­tion of pro­teins to fa­vor ni­tro­gen cap­ture, a par­tic­u­larly smart strategy of en­ergy real­loc­a­tion”, Wag­ner sums up. “Our next step will be to move into the mo­lecu­lar de­tails of the pro­cess and the en­zymes in­volved, as well as to look into other parts of the or­gan­is­m’s meta­bol­ism.”

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