Evidence of Martian Life Found in a Holland and Barretts?

With my looming redundancy date and seemingly receiving even more rejections than jobs I’ve applied for it was refreshing to get some good news this morning. We’ve had another paper accepted on our work on why answering the question “Is there life on Mars?” is actually really difficult (with current techniques).

This one has the typically short and punchy title of:'Transformation of cyanobacterial biomolecules by iron oxides during flash pyrolysis: Implications for Mars life detection missions'. Sadly I couldn't get my preferred title (see above) passed my co-authors...

It'll probably be a few weeks until it's officially published online but there's an open access preprint here.

Around 4.5-4 billion years ago Mars was less of a Red Planet and much more like the early Earth. At this point in time Mars still had a CO₂ rich atmosphere, replenished by volcanoes and protected by an active magnetosphere. This provided both a greenhouse effects and increased surface pressure which meant liquid water was stable at the surface. Rivers flowed and filled asteroid impact craters to form crater lakes which may have provided an environment quite hospitable to life.

Early Mars was probably a much nicer place than it is today

For this project I needed a life form that represented the sort of organism that may have evolved and colonised these environments, the remains of which could be preserved in the sort of lake sediments that NASA’s Curiosity has been exploring at Gale Crater, Perseverance is exploring at Jezero Crater, and ESA’s Rosalind Franklin may find at Oxia Planum in 2022.

What I needed was a simple, single-celled, prokaryotic photosynthesiser, capable of adapting to extreme environments. I needed a cyanobacteria, one form of which is the blue-green algal ‘scum’ which can cause poisonous blooms in ponds, while another can be bought from your local health food shop as a supplement to put in nutritious smoothies…

That’s right, we’re using Spirulina as a stand in for alien life.

Tasty smoothie or alien life?

In our last paper we looked at how, when Mars rovers heat up sediment samples any fatty acids present, which could be evidence of biological processes, will react with iron oxides and be transformed into highly ambiguous aromatic organic molecules which won’t look much like evidence of life at all. There's loads of background detail about the search for life on Mars in that post and others (including here, here and here) so I'll keep this brief and won't repeat it all now (I'm quickly writing this while procrastinating rather than doing interview prep).

This time round we wanted to make the system a little more complex by using whole bacteria (the Spirulina) to see if the same thing happened with intact, pristine biological matter. Unsurprisingly we found out that, yes, on heating the organic molecules released from the bacterial cells will still react with the iron oxides, transform into more ambiguous phases, and make it very difficult to be 100 % sure whether we’ve found life or not! The detected organic products look pretty much identical to those which could just have been delivered to the surface of Mars by meteorite impacts.

We did find some hope in that haematite, which is a common iron oxide on Mars, is nowhere near as problematic as the other iron oxides we studied, and that some biomolecules (isoprenoid hydrocarbons) had better survivability than others, handily these are also the molecules most likely to survive over the billions of years since Mars  was widely habitable. We also established that suites of molecules, which would be pretty much useless on their own, could in combination be relatively strong evidence of life – and at least be a good sign we should take a second look at the sample using a wider variety of techniques.

Graph of detected products from the Spirulina pyrolysed on the various iron oxide minerals, quartz used as a less reactive comparison. The original biomolecules (the blue, purple, green) which would be solid evidence of life are lost and transformed into more ambiguous species represented by the yellow, red and black. AS can be seen survivability of 'useful' biomolecules is higher at lower Spirulina concentrations in the haematite than other iron oxides (quartz as expected only has minor adsorption effects)

As with the last paper, we came to the conclusion that despite the problems, we shouldn’t try to avoid iron oxides altogether (this would be pretty much impossible on Mars anyway, iron oxides being everywhere are the reason it’s red). This is because iron oxides are both good for long term preservation of organic matter (it sticks to iron which offers some protection, known as the ‘rusty sink’ effect) and may indicate habitability as they are often formed in the presence of water and could provide an energy source for specialist microorganisms.

Instead, we should look for areas where any of the more problematic iron-bearing species will have been transformed to more amenable haematite. These are areas with evidence of long-lived (or shorter but hotter) oxidising fluid flow in the subsurface. These areas, such as Vera Rubin Ridge at Gale Crater and the clay units at Oxia Planum, have the added bonus of potentially acted as a refugia for life as conditions at the martian surface became more inhospitable. Bacteria may have retreated to them and eeked out an existence in the subsurface, ‘feeding’ on energy produced in chemical reactions for millions/billions of years after the water on the surface dried out.

Vera Rubin Ridge, as viewed by Curiosity, may be a good place to search for evidence of ancient life (credit NASA)

The next step of this research, already submitted, is to look at how ancient organic matter preserved over millions of years (in the form of kerogen) reacts with these problematic iron oxides.  It could be expected that this would to be more resistant to transformation as it has already lost much of its reactant groups but, spoiler alert, it’s not. Hopefully more details on that soon!

I should add that the publication of this paper also involved one of the best experiences of peer-review that I have had. Both reviewers clearly knew the topics and methods involved well and provided highly insightful comments picking up on mistakes and pointing out gaps in the manuscript and encouraging me to make it into a better, more impactful piece of work. I wish more reviewers were like this!

Has NASA been covering up knowing about Life on Mars since the 70’s?

Apologies for the clickbait title but the short answer is no.

However, this is something that came up a few times during our live Reddit Q & A session yesterday in the run up to Perseverance’s landing. I only had time to go into it briefly then so I thought I’d expand here for anyone who is interested in the details.

The real interesting question is: ‘Do some NASA scientists believe they found evidence of life on Mars in the 70’s and still think they’re being undeservedly ignored/silenced by the rest of the scientific community?’ Because the answer to that is a surprising yes.

Planning for the first landed missions to Mars started almost 70 years ago in the 1950’s. Back then it was thought that the surface of Mars was a much more benign environment than we know it to be today. Early observations of the planet seemed to suggest that there was seasonal vegetative cover and serious scientists (including Carl Sagan) postulated the idea of large martian animals roaming a landscape similar to Earth's in serious journals. It wasn’t until the Mariner Mars orbiter missions in the mid-late ‘60s that we got clear images back of the martian surface. While these showed it to be a more hostile, empty desert environment with no signs of animal or plant life, nor liquid water at the surface, the low resolution meant it did not preclude the existence of even macrofauna. Because of this, it was a real worry that microorganisms hitching a ride on Mars landers could invade the surface, outcompeting and leading to the extinction of any putative microscopic Martians, and wiping out our ability to ever know that life had ever existed on another planet.

This led to the Viking mission having the most intense planetary protection effort and the production of the cleanest, most sterile spacecraft, Viking 1 & 2, that have ever been launched. The expectation that we would find habitable surface conditions and martian microbes also led to one of the most interesting experiments ever carried out on another planet.

Viking lander, Carl Sagan for scale (credit: NASA)

The Labelled Release Experiments carried by both Viking landers were devised by Gilbert Leven and Patricia Straat. These experiments involved scooping up a sample of martian surface soil, ‘feeding’ it a nutrient broth labelled with radioactive isotopes and providing a little heat and water, then measuring any radioactive gas produced as the broth was ‘eaten’ by respiring martian microbes. On Mars, these experiments ‘worked perfectly’. When ‘fed’ the soil produced the radioactive gas exactly as would be expected if martian microbes were present. If the soil was first heat sterilised to kill any microbes present then less gas was produced as would be expected. The designers of the experiment and many others saw this as a huge success and clear evidence of life on Mars and published papers to that effect.

The first evidence that not all was as it seemed came quickly. The Viking landers were also equipped with instruments to analyse the martian soil to look for organic matter – which would be direct evidence of either the microbes themselves or their normal food source. Despite multiple attempts, no organic molecules were detected other than simple chlorinated molecules accompanied by large releases of carbon monoxide and carbon dioxide. This was problematic, a lack of organic matter in the soil meant no food for martian microbes to live on (when they weren’t being fed by us). This meant that very soon, most scientists preferred the hypothesis that the results of the Labelled Release Experiment could be caused by some strongly oxidising mineral, present in the martian soil, acting like a bleach to break down the nutrient broth, instead of the presence of martian microbes. At best the results were ambiguous. Levin and Straat disagreed, and have continued publishing support for their ‘life detection’ up to the present (Patricia Straat died in October 2020).

The mystery of the strong oxidant persisted for 40 years until the Phoenix lander detected perchlorate in the martian soil. Perchlorates and other oxychlorines have since been detected all over Mars by subsequent missions. Perchlorates are strongly oxidising chlorine containing salts. It is their presence that is believed to have caused the gas release measured in the Labelled Release Experiment as they oxidised the organic carbon ‘food’ in the nutrient broth on heating, mimicking biological activity. The presence of perchlorates also explains the lack of detection of organic matter by Viking’s thermal decomposition GCMS experiments. On heating in the sample oven, the perchlorates break down, releasing large amounts of oxygen and chlorine. This reacts with any organic matter present, causing it to combust and essentially burn away. This neatly explains the carbon dioxide and carbon monoxide, and the simple chlorinated molecules which were detected by Viking 1 & 2 and, more recently (since 2012), the Curiosity Rover.

Phoenix (credit: NASA)

So no, we haven’t yet detected evidence of life on Mars, the results of the Labelled Release Experiment can be more simply explained abiotically.

With everything we now know about the hostility of the martian surface we really don’t expect to find anything living there. The deep subsurface, sheltered from the harsh effects of UV radiation, solar and galactic rays, oxidising chemicals, extreme temperature fluctuations and aridity, is another matter entirely though. I would be very surprised if we never find evidence of at least extinct life on Mars, and am hopeful we will one day find weird microbes living deep beneath its surface.

Being the first step of eventual Mars Sample Return, planned for completion sometime in the 2030’s, the successful landing of NASA’s Perseverance rover yesterday will hopefully bring us one step closer to finally answering the question of whether there was ever life on Mars.

The first image sent back by Perseverance last night, minutes after landing (credit: NASA)

One final point for anyone who is not convinced about the lack of some great NASA conspiracy covering up life on Mars, and which also covers things like aliens visiting us, Oumuamua being an alien spaceship and flat Earth battshittery: If you saw the live coverage of the landing last night, or follow the Twitter accounts of the hundreds of NASA employees and thousands of scientists actively involved in these missions, you’ll see just how excited everyone gets and just how much they want to share EVERYTHING with the whole world. You really think these people could keep come massive discovery like that quiet? That’s just stupid...

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. 

The Nemesis Project

I’ve repeatedly heard that every academic, at some stage in their career, has that one project that just won’t behave itself and become a nice little publishable package. Experimental results lead to more questions than answers. Reviewers say the ides are interesting but are unconvinced by the conclusions or have issues in the reliability of the method. You're rejected but encouraged to re-submit. The whole thing drags on for years, quietly ticking away in the background, while research avenues with more promise for short-term gain are chased instead. But the nemesis project never dies, it stays there at the back of your head. Too much time and effort has already been invested, you’re in too deep to give up on it now.

My own mini-version of this has just ended. My nemesis project has just been published in Astrobiology (link, and link to non-paywalled pre-print) 3 years after my supervisor scribbled down a ‘cool idea which won’t take much time to test’ (I may be paraphrasing there, it was a long time ago). This is not my usual post-publication summary of my work, I will hopefully write that soon, instead this is a story of how much behind the scenes failure can have gone into one, small, relatively insignificant, successfully published paper.

The idea for the project came out of the PlanetaryProtection of the Outer Solar System project, which I have written about before (link). In one of the early meetings back in 2016 my supervisor, clearly paying full attention to whatever was being discussed at the time, scribbled a vague experimental idea onto a scrap of paper. This idea was to see if we could use a well-established environmental sampling technique (solid phase micro extraction, SPME) to test spacecraft hardware surfaces for organic contamination.  Now this was interesting as organic contamination is a big issue in planetary protection, we don’t want to send dirty spacecraft with highly sensitive instruments to the (currently) pristine icy moons of the outer solar system. We’d end up only detecting muck from Earth and so either getting all excited over nothing, misinterpreting it for evidence of alien life, or, a real interesting extra-terrestrial signal would be missed, lost in background noise from the contaminants. Current methods employed for detecting contaminants on surfaces tend to be time consuming, complicated and may involve multiple solvents being used in the process – themselves potential contaminants.

The premise was therefore simple: Get some stainless steel to use as a budget stand in for a spacecraft surface, contaminate it, see if SPME (coupled with GC-MS) is sensitive enough to detect contaminants at the levels of cleanliness required for life-detection missions.

The first version of this study just involved leaving some stainless steel L-shaped brackets (bought from a hardware store) out in the lab to collect fallout contamination from the air and also handling them with and without gloves to see if they picked up anything detectable from hand transfer.

To be scientifically valid a study like this must be reproducible, so many repetitions of everything being tested are needed, simple, but monotonous, time consuming work – perfect for an undergraduate summer internship! Georgios, a 2^nd year undergraduate and now co-author on the final paper, gave up 6 weeks of his summer in 2017 to do this, creating loads of data for me to work up afterwards. Now initially we thought this first version of the study was pretty good, however the reviewers had other thoughts.

Reject but encourage re-submission.

The issues basically boiled down to our method being a bit woolly and bullsh#t (again, paraphrasing). How could we know what was on the surface to detect and therefore how sensitive the method was if we hadn’t specifically contaminated it ourselves at a known concentration? We’d basically skipped the proof of concept stage and gone straight to real-word testing (well as real as you can get without a real spacecraft)...So yeah, fair enough.

Not having scope to dedicate 6 weeks of lab time to completely redo the experimental side of this study myself, the project had to get shelved until the following summer (2018) when I could get a second student, Yuting, who was keen to get some experience in the mind-numbing, soul destroying boredom of repetitive lab work.

In the meantime, I took the reviewer comments, which despite being rather critical were all very valid and helpful, and developed a whole new method for testing the sensitivity of this technique. This was to be much more scientific, creating a whole range of solutions of astrobiologically-relevant contaminants to contaminate a surface with much better-defined properties (although it was still basically just a steel nut).  

Once again, the student project seemed a success. Yuting produced a shed load of nice replicate data over the summer, which I turned into a completely new manuscript. None of the data set from Georgios’s original experimental run even made it into the new work, and after a few weeks of tidying up and writing we re-submitted.

Again, however, the reviewers didn’t quite agree, while they did think the method was now (mostly) sound, they didn’t agree the results were as promising as we did and wanted more and better data. Almost annoyingly this wasn’t a rejection this time, there was now a time pressure involved with a re-submission deadline. I could have ignored it and waited another year, but there was an end in sight, a way to kill this thing. Jon just needed to work some magic to tweak the mass spec settings to decrease the noise and make the data more convincing. Unfortunately, this meant I now had to repeat all the experimental work with the new settings myself, replicating a whole summer student project in about 3 weeks.

This was not fun, but it worked.

Now at the end of it all, it is clear that without the multiple knock backs and the intermediary time periods to just think about how to improve the methodology, this study would’ve been pretty rubbish. This is definitely a case where the review process has greatly improved an original idea and has shown me that rejections don’t always have to be a bad thing, they can be an opportunity. However, this only took 3 years to take down, I’m not sure how I’d feel if this had grown into some 5 or 10 year, or even career-spanning, monster.

Maybe that’ll be the next quick project…

Washing samples to detect 'Martian' organic matter

The beginning of this year seemed to be a good time for our group getting papers accepted and the third of those that snuck in is now online in Astrobiology:

Effects of Oxygen-Containing Salts on the Detection of Organic Biomarkers on Mars and in Terrestrial Analogue Soils. Wren Montgomery,Elizabeth A. Jaramillo, Samuel H. Royle, Samuel P. Kounaves, DirkSchulze-Makuch and Mark A. Sephton. (2019) Astrobiology

Once again, we’ve been looking at the troublesome effects of perchlorates on the detection of organic molecules, only this time we’ve used the Atacama Desert as a stand-in for Mars.

I’ve discussed the ‘perchlorate problem’ on Mars numerous times on this blog as it’s what I’ve been working on for the last couple of years (although not any more, watch this space). In short, oxygen-rich salts (of which perchlorate is probably the most problematic) are present in the Martian soil, we also expect there to be organic matter present (note, organic does not necessarily mean biological, see previous rant). With Mars lander missions, such as Curiosity, we attempt to detect the organic matter in the soil by heating the samples up in an oven to break down large molecules into smaller (more volatile) fragments that we can detect. However, those salts also break down when heated, releasing oxygen. This oxygen causes the organic matter to combust and any interesting organic molecules ‘burn up’ and are lost as carbon dioxide and carbon monoxide gases. This explains the difficulty that Mars missions have had in detecting organic matter on the surface, there has only been a single successful detection, which I’ll come back to later.

Mars today is a hyper-arid environment. While there may have been flowing rivers, lakes and even seas on the surface in the past, nowadays any liquid water anywhere near the surface is very rare. This allows the build-up of perchlorates and other salts which are highly soluble and would otherwise be washed away, which is why they are rare on Earth. This is where the Atacama Desert comes in as, recent floods notwithstanding, this is one of the driest places on Earth and so is one of the few places where perchlorates are present in the soil in significant amounts. This, combined with the low abundance of organic matter in the desert soil, due to its inhospitability to most life, makes the Atacama a (relatively) easy option for testing out ideas about Mars.

A rather Martian looking dawn in the Atacama Desert

The whole premise of this study can essentially be boiled down to: if these perchlorates are so soluble, can we just wash them out of our samples to allow us to detect the trace amounts of organic matter that we previously could not detect?

The answer, it turns out, is yes.

When the desert soil samples were initially analysed, by heating them in a similar fashion to what is carried out on Mars, showed little or no evidence of any organic matter being present.

Sub-samples of those soils were then well washed in very pure water, filtered and then dried. Unsurprisingly, analysis of the water showed that it had dissolved most of the soluble salts from the soils and it did not appear to have washed away any organic matter (which is mostly insoluble in water).

Once dried, analyse of these leached (washed) soil samples now allowed the detection of a variety of organic molecules. The molecules detected were indicative of the presence of cynobacteria (algae) that are known to be able to grow even in the dry desert.

This was, all-round, a pretty good result!

This potential for the problematic salts to be washed away has some pretty exciting implications for our search for organic matter on Mars. If we want to get around this ‘perchlorate problem’ we can either:

1. Wash our Martian samples with water. This, however, introduces a whole host of issues. Do we take water to Mars, it’s pretty heavy and we risk creating a nice, wet habitable environment for any Earth microbes that have hitched a ride, a major issue for planetary protection. Do we produce water on Mars by melting water-ice or extracting it from hydrated minerals, again, this would probably upset the mission’s Planetary Protection Officer (yes, this is a real job).

Buried ice exposed on steep slopes could be a useful water source

2. Go look for areas with evidence of ‘recent’ water activity on Mars were the salts will already have been leached away for us. This is the ‘easy’ option and what may have already happened accidentally. A rock unit called the lower Murray mudstone is the one place on Mars where evidence of complex organic matter has been found so far, co-incidentally this unit also has one of the lowest concentrations of perchlorate yet measured on Mars. There is evidence that, after the mudstones were deposited and buried, fluids flowed through the rock. These fluids could have leached away any soluble salts originally present, leaving being the insoluble organic matter, making it easier to detect. Areas with evidence of current or more recent water activity, such as above near-surface aquifers and near exposed and melting water-ice could also be promising areas to check out, however, these will also present planetary protection issues if there is liquid water available to support life.

Mineral veins show evidence of fluid flow, these fluids may have 'washed away' the soluble salts

This was actually a project that Wren had been trying to get published for a while now and the whole organic matter: perchlorate ratio paper we published last year actually originated as a response to reviewer’s comment to one of the early drafts of this current work. Happily, the two studies agree with each other, and NASA’S detection of organic matter which was announced while we were working on the re-write, pretty nicely (which is always good).