High-performance NMR spectrometer now in use for marine research

Dissolved Organic Matter is a mysterious and still largely unidentified combination of substances that is thought to be enormously important for the global climate. In Oldenburg, Germany, researchers are investigating this complex molecular mixture using two powerful instruments. An ultra-high-resolution mass spectrometer allows them to measure the molecular mass of the substances with a high degree of precision. The machine is one of the few of its kind in the world to be used in marine research. Now, the team has been equipped with a state-of-the-art NMR spectrometer, which will allow them to determine the exact molecular composition of the dissolved organic matter, and thus the processes that contribute to its formation and degradation.

Anyone who has had an MRI scan knows that things can feel pretty claustrophobic inside a tube with a diameter of just 60 to 70 centimetres. A machine with similar dimensions and based on the same physics principle was recently put into operation on the University of Oldenburg's campus, but inside there is only a few centimetres of room for measurements, and instead of human tissue the members of the Marine Geochemistry research group use their new nuclear magnetic resonance (NMR) machine to analyse the contents of sea water in thin glass tubes. "We're interested in organic molecules that are dissolved in seawater," explains Dr Phani Vemulapalli, a specialist in NMR spectroscopy. The team analyses seawater samples from all over the world, each litre of which contains a few milligrams of the organic material.

The mysterious and still largely unidentified combination of substances is thought to be of enormous importance for the global climate. Although the concentration of individual organic molecules in seawater is tiny, the total amount of organic matter in all the oceans combined is enormous: "Dissolved organic matter constitutes one of the largest carbon reservoirs on Earth, accounting for roughly the same amount of CO₂ as in the atmosphere," says Prof. Dr Thorsten Dittmar, who heads the Marine Geochemistry research group at the Institute for Chemistry and Biology of the Marine Environment. However, it is still not clear under what conditions this marine dissolved organic carbon reservoir expands or shrinks, thus influencing CO₂ levels in the atmosphere – or how it is reacting to climate change specifically. Dittmar, a geochemist, has spent years investigating these complex molecular mixtures. With the NMR spectrometer, he now has a new and powerful tool to advance his research.

A globally unique combination

Because Dittmar and his team can now finally analyse the structure of the organic molecules – in other words, how the atoms in a molecule are arranged and connected to each other. "This machine gives us a completely new perspective," says Dittmar. Since 2010, the team has been working with another highly sensitive machine, an ultrahigh-resolution mass spectrometer that can measure molecular mass with a high degree of precision and is one of the few of its kind in the world that is being used in the field of marine research. "Based on weight, we can measure the number of atoms of carbon, hydrogen, oxygen and certain other trace elements in a molecule," explains Dittmar. The mass spectrometer therefore makes it possible to determine and assign what are known as molecular formulas.

Dittmar and his team have identified around a hundred thousand different molecular formulas in water samples from all over the world in the last few years, but the total number of dissolved substances could be many times higher, because – similar to the letters in a word – for every molecular formula there are countless different ways in which the atoms may be arranged, or molecular structures. "There are millions of possible combinations, which is why we can't get any further with the mass spectrometer alone," Dittmar emphasises.

The team is now counting on the NMR spectrometer to shed light on the exact molecular composition of the Dissolved Organic Matter – and by extension, the processes that contribute to its formation and decomposition. The machine is based on the principle of nuclear magnetic resonance: in a powerful magnetic field, radio frequency radiation is used to make atomic nuclei absorb and emit energy at characteristic frequencies. These frequencies depend on the type of atom – carbon or hydrogen, for example – as well as on how the atoms are linked to each other. In this way, NMR spectroscopy provides an image of the atomic groups that make up a molecule. "To decode the extremely complex NMR signals, one needs cutting-edge computer programmes. It's like a huge three-dimensional puzzle," says Phani Vemulapalli.

NMR spectroscopy is already a standard procedure in chemical, biological, medical, and food sciences, but according to Dittmar it has rarely been used in environmental sciences as this field deals with highly diverse mixtures of organic compounds: "The signals from the individual molecules overlap, so we need extremely high resolution."

The new NMR machine can provide this resolution: it is one of the most powerful instruments of its kind. It cost 3.4 million euros, half of which was provided by the German Research Foundation through its major instrumentation funding programme and half by the state of Lower Saxony. Inside the machine, superconducting coils generate an extremely powerful magnetic field – around ten times stronger than in the average medical MRI scanner. It takes several hundred litres of liquid helium to cool the coils to minus 269 degrees Celsius – four degrees above absolute zero. Keeping the liquid insulated is also a complex process. "The helium chamber is surrounded by a chamber of liquid nitrogen and then a vacuum chamber on the outside," Vemulapalli explains.

"The pool of organic carbon compounds in the deep sea is far more dynamic than we thought"

The ICBM is currently the only institute in the field of environmental sciences to have the combination of a powerful NMR spectrometer and an ultrahigh-resolution mass spectrometer at its disposal – "a unique advantage", as Dittmar emphasises. "We are extremely fortunate to have such a measuring device," adds Vemulapalli, who previously conducted research at the NMR department of the Max Planck Institute for Multidisciplinary Sciences in Göttingen and is now contributing his expertise in Oldenburg. Access to this kind of equipment is limited and also expensive, which considerably limited the research of Dittmar's team in this field in the past.

Now the researchers have moved into uncharted territory – but they have already achieved unexpected results in their initial investigations. "The pool of organic carbon compounds in the deep sea is far more dynamic than we thought," says Dittmar. Previous analyses using the mass spectrometer indicated that the organic material in the different marine regions was several thousand years old and had a very similar composition across the globe. Based on this, the researchers had concluded that the carbon reservoir in the oceans was only changing very slowly. "However, the NMR measurements have shown that samples from different marine regions can contain very different molecules even if they share the same molecular formula," Dittmar reports. His hypothesis now is that, contrary to previous hypotheses, microbes use the mixture as food, but its growth and degradation rates are more or less in balance, so that the total quantity remains the same – "Like in a bathtub from which water flows out at the bottom while the same amount flows in at the top."

This is important information for global carbon cycle models such as those generated by biogeochemist Prof. Dr Sinikka Lennartz from the ICBM, because a lot depends on whether the dynamic equilibrium on our warming planet shifts. "The amount of carbon stored in the sea could increase, but it could also decrease," explains Dittmar. Lennartz has already incorporated the dynamic equilibrium into her ocean model. Results are expected in the near future.