Strange Isotopes: Scientists Explain a Mysterious Methane Isotope Paradox of the Seafloor

ROV Sampling Microbes

Sampling with the ROV in the home of the investigated microbes, the Guaymas-Beckens. Credit: Woods Hole Oceanographic Institution

Why methane carbon isotopes in the deep sea behave so differently than expected?

Deep down in the seafloor anaerobic microbes consume large amounts of methane, a potent greenhouse gas when it enters the atmosphere. Even though this process is a crucial element of the global carbon cycle, it is still poorly understood. Gunter Wegener from the Max Planck Institute for Marine Microbiology and the MARUM, Center for Marine Environmental Sciences, Bremen, Germany, and Jonathan Gropp from the Weizmann Institute of Science in Rehovot, Israel, now found the solution to a long-standing enigma in this process: why methane carbon isotopes behave so differently than expected. In a joint effort with their colleagues Heidi Taubner, Itay Halevy, and Marcus Elvert they present the answer in the journal Sci­ence Ad­vances.

Microbial ANME Consortium

Microbial consortia of anaerobic methane oxidizing archaea stained in red and their sulfate-reducing partner bacteria stained in green. The white scale bar marks 10 μm. Credit: Max Planck Institute for Marine Microbiology / V. Krukenberg

Meth­ane, a chem­ical com­pound with the mo­lecu­lar for­mula CH4, is not only a power­ful green­house gas, but also an im­port­ant en­ergy source. It heats our homes, and even sea­floor mi­crobes make a liv­ing of it. The mi­crobes use a pro­cess called an­aer­obic ox­id­a­tion of meth­ane (AOM), which hap­pens com­monly in the sea­floor in so-called sulfate-meth­ane trans­ition zones – lay­ers in the sea­floor where sulfate from the sea­wa­ter meets meth­ane from the deeper sed­i­ment. Here, spe­cial­ized mi­croor­gan­isms, the AN­aer­obic­ally MEth­ane-ox­id­iz­ing (ANME) ar­chaea, con­sume the meth­ane. They live in close as­so­ci­ation with bac­teria, which use elec­trons re­leased dur­ing meth­ane ox­id­a­tion for sulfate re­duc­tion. For this pur­pose, these or­gan­isms form char­ac­ter­istic con­sor­tia.

This process takes place glob­ally in the sea­floor and hence is an important part of the car­bon cycle. However, studying the AOM process is challenging because the re­ac­tion is very slow. For its in­vest­ig­a­tion, re­search­ers of­ten use a chem­ical knack: the stable iso­tope ra­tios in meth­ane. But un­for­tu­nately, these iso­topes do not al­ways be­have as expected, which led to ser­i­ous con­fu­sion on the role and func­tion of the mi­crobes in­volved. Now re­search­ers from the Max Planck In­sti­tute for Mar­ine Mi­cro­bi­o­logy and the MARUM – Cen­ter for Mar­ine En­vir­on­mental Sci­ences in Ger­many to­gether with col­leagues from the Weiz­mann In­sti­tute of Sci­ence in Is­rael have solved this iso­tope en­igma and pub­lished their res­ults in the journal Science Advances. This paves the way for a bet­ter un­der­stand­ing of the im­port­ant pro­cess of an­aer­obic meth­ane ox­id­a­tion.

Iso­topes re­veal re­ac­tion path­ways

The puzzle and its solu­tion in de­tail: Iso­topes are dif­fer­ent “ver­sions” of an ele­ment with dif­fer­ent masses. The iso­topes of an ele­ment have the same num­ber of pro­tons (pos­it­ively charged particles) in the nuc­leus and therefore the same po­s­i­tion in the peri­odic table (iso topos = Greek, same place). However, they dif­fer in the num­ber of neut­rons (neut­ral particles) in the nuc­leus. For example, car­bon has two stable iso­topes, the lighter 12C and the heav­ier 13C. Ad­di­tion­ally, there is the fa­mil­iar ra­dio­act­ive iso­tope 14C, a very rare car­bon spe­cies that is used to de­term­ine the age of car­bon-bear­ing ma­ter­i­als. Although the chem­ical prop­er­ties of the two stable iso­topes are identical, the dif­fer­ence in mass res­ults in different re­ac­tion rates. When chem­ical com­pounds re­act, the ones with the lighter iso­topes are usu­ally con­ver­ted faster, leav­ing the heav­ier vari­ant in the ini­tial re­act­ant. This change in iso­topic com­pos­i­tion is known as iso­topic frac­tion­a­tion and has been used for dec­ades to track chem­ical re­ac­tions. In the case of meth­ane ox­id­a­tion, this means that 12C-meth­ane is primar­ily con­sumed, lead­ing to an en­rich­ment of 13C in the re­main­ing meth­ane. Con­versely, a mi­cro­bial pro­duc­tion of meth­ane (meth­ano­gen­esis) would res­ult in par­tic­u­larly light meth­ane. “Real­ity, however, is sur­pris­ingly dif­fer­ent,” Gunter We­gener re­ports. “Con­trary to the lo­gic de­scribed above, we of­ten find very light meth­ane in sulfate-meth­ane trans­ition zones.”

Guaymas Basin Hydrothermal Vents

The Guaymas Basin hydrothermal vents – the “home” of the studied methane-oxidizing microorganisms. The heat-loving microorganisms thrive under the orange microbial mat in the background. The high temperatures of the rising waters blur parts of the image. Credit: Woods Hole Oceanographic Institution

Nature does­n’t fol­low the text­book: Light meth­ane in sulfate-meth­ane trans­ition zones

This para­dox raises questions, such as: Is meth­ane not con­sumed there, but rather pro­duced? And who, if not the nu­mer­ous ANME ar­chaea, should be re­spons­ible for this? “In my lab, we have the world’s largest col­lec­tion of ANME cul­tures. There we could try to find out if and how the meth­ane ox­id­izers them­selves could be re­spons­ible for the form­a­tion of light meth­ane,” We­gener con­tin­ues. “The first res­ults were de­flat­ing: At the high sulfate con­cen­tra­tions we nor­mally find in sea­wa­ter, the cul­tured mi­croor­gan­isms be­haved ac­cord­ing to the text­book. The re­main­ing meth­ane was en­riched in the heav­ier iso­topes.” However, if the same ex­per­i­ments were carried out with little sulfate, meth­ane got en­riched in 12C, it became lighter. And this happened even though meth­ane con­tin­ued to be con­sumed at the same time – an ef­fect that at first glance had little lo­gic.

The avail­ab­il­ity of sulfate gov­erns the iso­topes ef­fects in AOM

So how could they ex­plain the un­usual be­ha­vior of the meth­ane iso­topes? Jonathan Gropp and his mentor Itay Halevy from the Weiz­mann In­sti­tute of Sci­ence in Is­rael have spent years study­ing the iso­tope ef­fects of mi­cro­bial meta­bol­isms, in­clud­ing meth­ano­gen­esis – a re­ac­tion that is cata­lyzed by the same en­zymes as the an­aer­obic ox­id­a­tion of meth­ane (AOM). Thus, they were the ideal part­ners for the team located in Bre­men. “Both pro­cesses are based on a very sim­ilar cas­cade of seven re­ac­tions,” says Gropp. “Pre­vi­ous stud­ies have shown that all of these re­ac­tions are po­ten­tially re­vers­ible, meaning that they can take place in both dir­ec­tions. Each re­ac­tion also has its own iso­tope ef­fects.” With the help of a model, Gropp was able to show that, de­pend­ing on how much sulfate is avail­able, the par­tial re­ac­tions can be re­versed to vary­ing de­grees. This could then lead to the situ­ation that heavy iso­topes are not as usual left behind but are stuck in the re­ac­tion chain, while light iso­topes are channeled back to meth­ane. “The mi­crobes want to per­form the re­ac­tion but are lim­ited to do so be­cause of the low sulfate con­cen­tra­tions,” explains Gropp, adding that “Our de­signed model fits the iso­tope ex­per­i­ments very nicely.”

The long hours in the labor­at­ory and in front of the com­puter paid off for the re­search­ers. With their study, We­gener, Gropp, and their colleagues could show how AOM res­ults in 13C-de­pleted meth­ane. The ex­per­i­ments with little sulfate in par­tic­u­lar nicely re­flect the con­di­tions in the nat­ural hab­itat of the mi­croor­gan­isms, the sulfate-meth­ane trans­ition zones in the sea­floor. There, the mi­croor­gan­isms of­ten thrive on only little sulfate, as in the low-sulfate ex­per­i­ments. “Now we know that meth­ane ox­id­izers can be re­spons­ible for the build-up of light iso­topes in meth­ane at sulfate-meth­ane trans­ition zones. Meth­ano­gen­esis is not re­quired for that. As we sus­pec­ted, the ANME are meth­ane ox­id­izers,” con­cludes Mar­cus Elvert, last au­thor of the cur­rent study. Now the re­search­ers are ready for the next step and want to find if other re­ac­tions show sim­ilar iso­tope ef­fects.

Reference: “Sulfate-dependent reversibility of intracellular reactions explains the opposing isotope effects in the anaerobic oxidation of methane” by Gunter Wegener, Jonathan Gropp, Heidi Taubner, Itay Halevy and Marcus Elvert, 5 May 2021, Science Advances.
DOI: 10.1126/sciadv.abe4939

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