Paper Summary: Pyrolysis of carboxylic acids in the presence of iron oxides: Implications for life detection on missions to Mars

Normally I wouldn't even dream of doing any work 2 days before Christmas, but this year is a bit different. We're stuck in London's Tier 4 Festive Hell and Charlotte and I ran out of conversational topics half way through June, so I thought I'd share this tiny bit of good news at the end of a terrible year, we’ve had another paper accepted! This one was initially submitted all the way back at the end of 2018 before we’d even heard of social distancing or Barnard Castle. I remember because it received its first rejection while I was at a real in-person conference (who knows when they’ll be back) in Scotland (we’re never allowed back there are we?).

The new paper is the snappily titled Pyrolysis of carboxylic acids in the presence of iron oxides: Implications for life detection on missions to Mars. It is the second in a series of papers we’re hoping to get out looking at how the presence of iron containing minerals may have affected attempts to detect organic matter, including life detection efforts, on Mars. You can read an open access version here.

I’ve discussed what organic matter is and why efforts to find life on Mars revolve around its detection on here before. But briefly, organic matter is made up of organic molecules, these are chemical compounds that contain carbon (C) and hydrogen (H). The C-H backbone is very flexible and reactive but also manages to be quite stable. This allows many different elements and functional groups to join on, in various positions and shapes, to form a very wide range of carbon-based molecules, some of which may be quite large and complex. Organic molecules can form through just the action of heat or radiation on inorganic carbon-bearing species (like carbon dioxide), even in the depths of space. But the unique properties of organic molecules allows carbon-based chemistry to form the building blocks of all known life; as biology takes simple organic molecules and uses them to build complex biomolecules. Many non-biological processes can synthesize surprisingly complex organic molecules, however, certain structures and patterns are almost statistically impossibly to be formed by random chemical interactions, they may only be produced by the dedicated, enzyme controlled processes of life. If preserved in sediments, these biological structures are known as organic biomarkers and, as pretty solid evidence for life, their detection would be the ‘smoking gun’ of Mars life detection efforts.  

We may expect to find biomarkers on Mars as around 3 to 4 billion years ago, around the same time life was evolving on Earth, Mars was a much more pleasant place to be. It was warmer and wetter as it still had an atmosphere, replenished by volcanic activity and protected by a stronger magnetic field. There were rivers, lakes and even oceans. All of the ingredients for life to evolve were present for millions, if not hundreds of millions, of years. If life did evolve it would leave its molecular fossils, biomarkers, in the sediments for us to detect today. Even if life didn’t evolve, there should still be evidence of interesting prebiotic chemistry occurring due to hydrothermal or magmatic processes (as we do see evidence of this in martian meteorites).

Landed Mars missions have yet to find any definitive biomarkers, although they have detected a suite of small, simple organic molecules that appear to be the fragmentation products of larger molecules (a macromolecule), and some longer chain alkanes which have been suggested to be the breakdown products of fatty acids. All attempts to find organic molecules on Mars so far have used methods that rely on heating up samples to break down and volatilise organic matter into smaller fragments that can be separated, detected and identified. The problem is that that also heats up any inorganic minerals that are also present, and make up the bulk of, the sample. Some of these minerals may be highly oxidised and release oxygen on heating, essentially burning up any organic matter that is also present in the sample. In the best case scenario this organic matter becomes overly fragmented and loses structural information diagnostic of its source, in the worst case it is totally lost to analysis as it oxidises to carbon dioxide and carbon monoxide. This is what has been blamed for the lack of conclusive detections so far as we know that some salts (particularly perchlorates) have had this effect.

Another factor that has so far been less explored is the effect of other, 'less reactive', inorganic minerals in the samples. Iron oxides are widespread across the surface of Mars, they’re the reason it is the Red Planet after all. Therefore, we wanted to look at the effects iron oxides could be having on attempts to find and identify biomarkers in the martian sediments. We had already had some clues that iron oxides may affect biomarker detection from Jonny’s work that was published a few months ago. This showed that in natural samples that were rich in both iron and organic matter, you had to remove the iron-bearing minerals to be able to properly detect the organic molecules and fully identify the source of organic matter.

Mars is the Red Planet due to iron oxides at the surface (credit: NASA)

To work out exactly what was going on we needed a MUCH simpler system than Jonny’s stream environment, so we made our own analogue samples to eliminate unknown variables.

We decided to use 2 mid-long chain length fatty (carboxylic) acids, both containing 18 carbon atoms but in different saturation states.  Oleic acid, an unsaturated fatty acid that is a major component of vegetable oils, and stearic acid, a saturated fatty acid that is found in many animal and vegetable fats. In this context unsaturated and saturated mean whether the molecule contains any double carbon-carbon bonds or not, which is the same meaning as when you talk about fats in food. Fatty acids are useful molecules to look at as their chain length and saturation state can provide a lot of information about their probable source: biological processes select for longer chain lengths whereas non-biological processes are statistically more likely to produce shorter chain lengths and unsaturated molecules saturate over time, so a concentration of longer, unsaturated fatty acids may be a good indicator of recent life.  

Steric acid (left) and Oleic acid (right)

We mixed these fatty acids into a variety of inorganic minerals, we used quartz as a ‘control’ sample, as this mineral is known to not be very reactive, and tested the iron oxides haematite and magnetite; the iron oxyhydroxide goethite and the iron hydroxide ferrihydrite. All of these minerals have been directly detected or inferred to be present at the surface of Mars.

We then analysed these fatty acid-mineral mixtures in a way similar to that which is used to analyse samples at Mars; by heating them up in an inert atmosphere and seeing what organic molecules were released, a technique called pyrolysis-gas chromatography-mass spectrometry.

What we observed was that on heating the organic matter and iron-bearing minerals reacted with each other. This altered the organic products detected as the breakdown of the fatty acids was enhanced and the products were transformed into other species, far less diagnostic of their source. This led to a reduction in both the abundance and variety of products detected, especially when lower (more realistic) concentrations of fatty acids were used. A serious loss of diagnostic structural information meant that the products of these fatty acids, which could have been indicative of life, were pretty much indistinguishable to the expected breakdown products of abiotic, mature macromolecular matter. Abiotic macromolecular organic matter is what has already been inferred to have been detected at the surface of Mars and is the sort of thing we would expect to detect there as it could be delivered by meteorites (they’re full of the stuff).

Iron oxides promote the fragmentation & transformation of fatty acids into molecules more normally indicative of abiotic macromolecular matter
 

The inability to distinguish between low concentrations of ‘fresh’ biologically derived molecules and ancient abiological matter in the presence of iron minerals is a serious problem for life detection efforts at Mars, however our work did suggest a potential solution. Quartz had very little effect on the breakdown of the fatty acids, however, quartz-rich sediments are not a good environment for preserving organic matter over geological time as they are actually not reactive enough, iron-bearing minerals are much better because the organic matter binds to its surface, providing some protection. Out of all the iron-bearing minerals we tested the ‘least bad’ was haematite, this means that, on Mars, we should be looking for organic matter in sediments that have been subjected to conditions where haematite is the most stable form of iron. Haematite is the most stable iron-bearing phase under oxidising and acidic conditions, especially at higher temperatures, and there are numerous localities on Mars where we have evidence (from mineral veins) that hydrothermal fluids fitting this description flowed through the rocks while they were buried. At these localities, any of the more reactive iron-bearing phases will have already been replaced by haematite, which based on some of the experiments Jonny did for his PhD thesis, shouldn’t negatively affect the preservation of any organic matter adsorbed onto those minerals.

Veins provide evidence of hydrothermal fluid flow 

So, in conclusion, iron oxides are going to be problematic for the detection and identification of fatty acids on Mars. However, as they are good for preservation of organic matter over geological time periods we can’t just avoid them. Instead we have to target localities where haematite is the most stable iron oxide as this seems to be the ‘least bad’.

I am currently in the process of submitting a follow-up paper examining what effect these iron-bearing minerals have on the detection of biomarkers from whole bacteria and we’re also looking at a few other Mars-relevant sources of organic matter. Watch this space but they seem to cause very similar problems for detecting those as well…

Merry Christmas!

Paper Summary: Artificial maturation of iron- and sulfur-rich Mars analogues (AKA Is There Life in Dorset?)

Artificial maturation of iron- and sulfur-rich Mars analogues: Implications for the diagenetic stability of biopolymers and their detection with pyrolysis gas chromatography–mass spectrometry (Tan, Royle and Sephton, Astrobiology, 2020) 

AKA Is There Life On Mars In Dorset? 

Gale Crater, Ancient Mars/ St Oswolds Bay, Dorset; the similarities are remarkable.... 

So despite the global pandemic shutting down the lab and having to spend weeks in a Singaporean quarantine centre with coronavirus himself, Jonny has managed to get the lab’s only 2020 paper accepted. It’ll be a few weeks until the final version is online but an open access pre-proofed version of the accepted manuscript can be found here (link). As it’s a quite a long paper and we’re all busy trying to survive the end times, we’ve tried to write a accessible summary here to get the main points across…. 

Around 4-3.5 billion years ago (the Late Noachian to Hesperian periods) the surface of Mars was a much more habitable place than it is today. Increased volcanic activity and a protective active magnetic field maintained Mar's atmosphere. This provided a global warming effect, allowing liquid water to be stable on the martian surface (at least some of the time). Rivers flowed, valleys were formed, and lakes were filled. This is around the same time that life evolved on Earth, and if it also evolved on Mars, or hitched a ride there from Earth via meteorite, it may well have flourished under these conditions. Therefore, our best chance to find evidence of ancient martian life will be in the sediments deposited in this period. 

As well as providing an atmosphere, those volcanoes injected large volumes of sulphur dioxide (SO2) into the atmosphere and made the waters quite acidic. This encouraged the deposition of sedimentary sulphate minerals and iron oxides. On Earth, acidic groundwaters containing dissolved sulphates bubble up in a few places to produce sulphur streams and precipitate sulphate salts, such as jarosite, alongside iron oxides, such as haematite and goethite. These streams provide handy analogues for ancient habitable martian environments, especially as some can be found as nearby as Dorset! 

In a previous paper (link), Jonny already established that organic biosignature molecules (the ‘fingerprints’ of life) are concentrated within the iron-rich phases of the sulphur stream environment and that they may be detected by techniques similar to the capabilities of current and future Mars Rover missions. 

The new work takes this a step further, to see what would be detectable after the nearly 4 billion years that have passed since this most habitable period of Mars. During that time the sediments will have been buried, heated and subsequently uncovered; any sediments that have not been buried on Mars will have had all their interesting organic molecules destroyed by cosmic and solar radiation so there's no point looking in those. We know that the sediments at Gale Crater that Curiosity has been poking at were at one point buried to at least 1.2 km depth. Increased pressure and temperature, when coupled with potentially reactive mineral surfaces, may destroy or at least alter, the evidence of life (organic biosignature molecules) we are searching for. 

Sediment samples were collected from 2 sulphur streams in Dorset, at St. Oswald’s Bay and Stair Hole. These sites are known to be inhabited by weird extremophile microbial life, including acidophilic (acid loving) algae and microbial mats of phototrophic purple sulphur bacteria (they use sunlight to make energy out of sulphur), which thrive in these harsh conditions. 

The sulphur stream, green acidophillic algae and purple sulphur bacteria are easy to spot so should be easy to detect their 'biosignatures'

After being freeze-dried, these samples were artificially matured using hydrous pyrolysis. In this technique millions to billions of years of burial and low temperature heating can be replicated within a few days by using higher temperatures to speed up the reactions (Arrhenius equation). After this any surviving soluble organic matter was extracted with solvents, as in reality this would be lost due to the actions of percolating fluids through the sediment, so we only want to look at the insoluble fraction left behind. To see what effects the sulphates and iron oxides in the sediments had on the detectability of the organic material during the analysis step these were removed using strong acid and alkali washes to dissolve them away for half the samples.

 'Bomblets' and pressure vessel 'bomb' used for hydrous pyrolysis, billions of years of burial in one weekend!

After all this preparation, the samples were analysed by pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS). This technique is similar to the main way that all Mars missions have looked for organic matter in martian sediments/rocks. By heating up the sample organic matter is liberate and volatilised into fragments that can be separated and identified (more details here). This technique, whilst being the simplest way of detecting insoluble macromolecular material (of the type we may expect to be left behind after billions of years of burial) the heating encourages reactions between the organic matter and reactive/oxidising mineral surfaces, which has been a problem in the past (hopefully we’ll have a paper looking at this in more detail out very soon); hence the acid/alkali treatment to remove these phases in this study. 

Unsurprisingly, the sulphur stream samples that had not been artificially matured were found to produce a wide range of organic compounds, consistent from those generated from the pyrolysis of microbial mats from similar environments. The shear amount of organic matter in the sample was able to overcome any issues in detection due to the presence of the sulphates and iron oxides in the samples. However, samples that received the acid/alkali wash still produced a greater abundance and variety of organic molecules than those which did not. Many of the organic compounds detected are diagnostic of biology and have the potential to be used as biosignatures. Some can be related to bacteria, with markers of both anaerobic and aerobic metabolisms present, while others indicate an input of woody higher plant material. 

Pyrolysis-GCMS data showing what organic molecules may be detected from the same sulphur stream sample with different pre-treatment regimes, before and after hydrous pyrolysis and before and after acid treatment 

After artificial maturation/diagenesis, organic matter detectability decreased markedly with increased hydrous pyrolysis temperature as organic matter was degraded. It was not until the sulphates and iron oxides were dissolved away that we could see anything interesting, i.e. nothing diagnostic of life was detected without the acid/alkali treatment! After they were removed it could be observed that many biosignature compounds did, in fact, survive the maturation process, although some were lost or altered.

  

So, why’s this interesting at all? Well it demonstrates that if we only use bog standard thermal decomposition (pyrolysis) techniques to look for organic matter in martian sediments which are rich in sulphates and iron oxides then we’re going to miss out on a lot of stuff, we just won’t see biosignatures that are there! Sulphates and iron oxides are yet another barrier to organic matter detection by thermal extraction strategies and should be avoided where possible. Issues with this may have already happened, the simple organic compounds detected by Curiosity in 2018 (link) look rather similar to what was detected in the un-acid/alkali washed samples and were detected in mudstones with high sulphate and iron oxide contents! If these reactive minerals could have been removed in a pre-treatment step prior to analysis, who knows how much of a greater variety of organic information would have been unlocked, perhaps even the first compelling biosignatures of ancient life on Mars? 

Simple organic compounds already detected on Mars, but what information was lost due to reactive minerals?

Sorry Venus, Mars is still where it's at!

 All of the excitement in the astrobiological community of late has been on the detection of phosphine in the clouds of Venus and how this may be a ‘biosignature’ of microbial life in the ‘habitable’ environment of the Venereal (pretty sure that’s the correct term…) clouds. While this detection is pretty damn cool, I do think the detection of a simple molecule that can also be produced from volcanoes and lightning (which we know exist on Venus) or from some other weird high temperature geochemistry (we know sod-all about Venus really) is getting a little over-hyped (in the same vein as Curiosity’s 2018 ‘life’detection).   

Can always rely on the Daily Express...

Now what is worth getting excited about is the Curiosity rover finally carrying out a TMAH experiment this month (after nearly 8 Earth years on the martian surface). For non-organic geochemists; TMAH, or tetramethylammonium hydroxide, is one of the two derivatisation agents carried by SAM (Sample Analysis at Mars, Curiosity’s onboard chemistry laboratory), the other being the even more god-awfully named MTBSTFA or N-methyl-N-(tert-butlydimethylsilyl)trifluoroacetamide.

Thanks to previous experiments by Curiosity, we now know that organic molecules are certainly present on Mars, they may exist as complex macromolecules, and their detectability is affected by the presence of various minerals on the martian surface. The usual technique for detecting organic matter on Mars, thermal decomposition (pyrolysis), is a bit of a blunt instrument, as using heat to release organic molecules from the sediments basically blows them apart (especially in the presence of oxidising salts), destroying structural information and making it difficult to establish their provenance. Because of this, so far, we have only detected simple organic molecules on Mars with much speculation, but little evidence, to their source (reminder – organic molecules, whilst they are the building blocks of life, they can also be produced by many abiotic processes). In contrast, derivatization agents are a really useful tool in detecting and understanding organic molecules as they can liberate organic molecules of interest from macromolecular matter or (potentially reactive) mineral surfaces less destructively and at lower temperatures.

A particularly interesting class of molecules in the search for life on Mars are the fatty acids. Unlike most other (potential) biomolecules fatty acids survive well under harsh environmental conditions over geological timescales (pretty important as Mars’s most habitable conditions were over 3.5 billion years ago) and can contain much information suggestive of their source. Fatty acids, as the name suggests are the main breakdown products of lipids or fats. Abiotic processes (such as hydrothermal processes) primarily produce short fatty acid molecules, whereas life tends to use longer fatty acid chains with even carbon numbers as essential components of cell membranes. The specific lengths and saturation states of these longer fatty acids can also provide clues as to the type of life they came from, bacteria, algae, higher plants all leave behind specific distribution patterns of fatty acid chain lengths. Hopefully I'll be posting a more in-depth discussion of why fatty acids are a good target in the search for martian life shortly as we're trying to get a couple of papers published on the subject...

Oleic acid, an 18 carbon singularly unsaturated fatty acid

However, these potential biosignatures are notoriously tricky to detect as fatty acids (a) are ‘sticky’ and so are hard to get off mineral surfaces in the first place; (b) on heating they break down pretty easily to ‘boring’ alkenes/alkanes which don’t preserve much information about their source; and (c) even if you liberate them from the mineral surface intact they are a polar molecule so the gas chromatograph-mass spectrometer’s (GCMS) detectors only ‘see’ them if present in large quantities (unlikely on Mars).

If a TMAH derivatisation step is applied to the samples before pyrolysis, however, the fatty acids can be liberated from the macromolecules/mineral phases at a lower temperature. Heating in the presence of TMAH hydrolyses organic matter, freeing the fatty acids (and other bound molecules) and also methylates (adds a methyl -CH₃) to polar functional groups, including the carboxylic group of the fatty acid molecule. The methylation makes the fatty acid (or other polar molecules) less polar and more volatile, making them more amenable to detection in the GCMS. Here's an open access paper on this technique being used on Mars analog samples if you want (significantly) more detail.

The easier to detect methyl ester of oleic acid

This is exciting as this experiment will be our best chance so far of detecting more complex organic molecules, work out where they are from and ultimately find evidence of life on Mars (maybe). Unfortunately we'll have to wait months to find out the results as the scientists involved will have to carry out all sorts of experiments to validate the rover's findings (especially if they find something that looks particularly interesting).

Further into the future, the ExoMars Rosalind Franklin rover, scheduled for launch in 2022, will have the ability to carry out other derivatisation pyrolysis experiments which work at temperatures down to 250 or 140 depending on the exact technique used. It will also have a laser desorption unit which will liberate organic molecules through millisecond laser pulses, a non-destructive technique. Both of these abilities should preserve more structural information and be less likely to suffer mineral surface effects than any experiments we have been able to do on Mars with Curiosity or any of the previous landed missions.