Amy J Williams
email: amywilliams1@ufl.edu

Kathleen L Craft

Maëva Millan

Sarah Stewart Johnson

Christine Knudson

Marisol Juarez Rivera

Amy C Mcadam

Dominique Tobler

John Roma Skok

Mary Ann Liebert

Sarah Stewart Johnson

Knudson

Mary Ann

Fatty Acid Preservation in Modern and Relict Hot-Spring Deposits in Iceland, with Implications for Organics Detection on Mars

Keywords: Hot Springs, Organic Geochemistry, Mars, Analog, Biosignatures hot springs, sinter, FAME, MSL, Sample Hot springs, Sinter, Fatty acids, TMAH, SAM instrument

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

 GC-MS (del Rio et al., 1996;Metcalffe and Wang, 1981), as well as freeing bound 119 components of larger macromolecules otherwise undetectable with GC-MS [START_REF] Grasset | Mechanisms of thermal adaptation in bacteria: blueprints for survival[END_REF]. ), thermochemolysis with TMAH allows the analysis of refractory matter in complex 131 matrices containing low amounts of organic material with minimal destruction of the organic 132 material [START_REF] Geffroy-Rodier | Thermochemolysis 859 in search for organics in extraterrestrial environments[END_REF]He et al., 2019;[START_REF] Williams | Recovery of Fatty Acids from 1096 Mineralogic Mars Analogs by TMAH Thermochemolysis for the Sample Analysis at Mars 1097 Wet Chemistry Experiment on the Curiosity Rover[END_REF]. TMAH 133 thermochemolysis selectively cleaves ester and ether bonds, replacing a labile hydrogen atom with 134 a methyl group (Figure 2), rather than random and uncontrolled thermal decomposition and 135 fragmentation of the organic material (as during pyrolysis). TMAH has been used for several 136 decades in a variety of terrestrial fields of study, including petroleum geochemistry (Larter and 137 Horsfield, 1993), soil science (Chefetz et al., 2000;Deport et al., 2006;[START_REF] Schulten | Direct pyrolysis-mass spectrometry of soils: a novel tool in agriculture, 1029 ecology, forestry and soil science[END_REF], and 138 sedimentology (Guignard et al., 2005;Pulchan et al., 1997). TMAH is currently available on the 139 Curiosity rover SAM instrument (Mahaffy et al., 2012;[START_REF] Williams | Recovery of Fatty Acids from 1096 Mineralogic Mars Analogs by TMAH Thermochemolysis for the Sample Analysis at Mars 1097 Wet Chemistry Experiment on the Curiosity Rover[END_REF] and will also be used 140 on the ExoMars rover MOMA instrument (Goesmann et al., 2017). Fortunately, perchlorate does Yellowknife Bay, Gale crater (Freissinet et al., 2015), and thiophenic, aromatic, and aliphatic 148 organic compounds were found in the 3.5 Gy Murray Formation lacustrine mudstones of Pahrump 149 Hills, Gale crater (Eigenbrode et al., 2018). In more recent SAM experiments, preliminary analyses 150 suggest the presence of select medium-chain alkanes [START_REF] Williams | Recovery of Fatty Acids from 1096 Mineralogic Mars Analogs by TMAH Thermochemolysis for the Sample Analysis at Mars 1097 Wet Chemistry Experiment on the Curiosity Rover[END_REF] and medium to high 151 molecular masses, including derivatized molecules [START_REF] Williams | Recovery of Fatty Acids from 1096 Mineralogic Mars Analogs by TMAH Thermochemolysis for the Sample Analysis at Mars 1097 Wet Chemistry Experiment on the Curiosity Rover[END_REF], in samples exposed to 152 SAM's other wet chemistry experiment, derivatization with MTBSTFA (Buch et al., 2006).

153

These findings represent the first confirmed in situ organics detection on Mars, but the source of 154 these organic molecules is uncertain. Abiotic Martian sources (e.g., from atmospheric, 155 hydrothermal, or igneous processes), abiotic exogenous sources such as comets, interplanetary 156 dust particles, or meteorites (Eigenbrode et al., 2018;Freissinet et al., 2015), and life all produce 157 organic molecules. TMAH thermochemolysis, however, provides an unprecedented opportunity 158 to assess the biogenicity of Martian organics, specifically carboxylic and fatty acids. Fatty acids, 159 bound in phospholipids and glycolipids, are abundant and ubiquitous components of eukaryotic 160 and bacterial cellular membranes [START_REF] Vestal | Lipid Analysis in Microbial Ecology[END_REF]. Carboxylic acids have also been >C 30 in length have an even-over-odd carbon chain length preference due to enzymatically formed 165 acetyl (C 2 ) units derived from glucose [START_REF] Volkman | Lipid biomarkers for marine organic matter[END_REF]. Abiotic fatty acid patterns will favor 166 shorter carbon chain lengths with no carbon preference (Bray and Evans, 1961;McCollom et al., 167 1999), although it is important to note that very short chain carboxylic acids can represent 168 metabolic byproducts that were not incorporated into cellular membranes. Therefore, within a 169 certain level of uncertainty, characteristics of a FAME profile could be used to deconvolve the 170 origin of fatty acids detected on Mars [START_REF] Williams | Recovery of Fatty Acids from 1096 Mineralogic Mars Analogs by TMAH Thermochemolysis for the Sample Analysis at Mars 1097 Wet Chemistry Experiment on the Curiosity Rover[END_REF].

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The work presented here investigates the preservation and detection of organic molecules,

202

A second site, Gunnuhver, lies on the Reykjanes peninsula in SW Iceland (Figure 3C). The 4).

208

The third site visited was Lýsuhóll, a farmland area with relict sinter deposits and few active vent 209 systems (Figure 3D) with lower temperatures (ca. 41°C) and pH = 6.3. Other low temperature (ca. were sampled (Figure 4).

216

All samples were collected with the appropriate permissions. Hveravellir is a protected natural 217 monument for which we were permitted to sample by the Environment Agency of Iceland. We or subsampled in the lab from the solvent washed and ashed glass jars with solvent washed tools.

233

In the laboratory, sinter from the glass jars was subsampled by breaking off smaller pieces with 234 solvent washed and ashed tweezers. These subsamples were powdered with a solvent washed and reflecting more extant diverse microbial communities.

363

Most All samples contain FAMEs longer than n-C 18:0 , which most often represent a terrigenous 364 plant or algal origin [START_REF] Volkman | Fatty acid and lipid 1070 composition of 10 species of microalgae used in mariculture[END_REF]Kaur et al., 2011;[START_REF] Wilhelm | Xeropreservation of functionalized lipid biomarkers in hyperarid soils in the Atacama 1091 Desert[END_REF]Johnson et al., 365 2020), although these fatty acids may be derived from bacterial free, long chain fatty acids 366 [START_REF] Summons | Lipid 1054 biomarkers in ooids from different locations and ages: evidence for a common bacterial 1055 flora[END_REF] or intact polar lipids. Based on the abundance of surface vegetation near 367 the spring sites, the FAMEs here are assumed to represent waxy longer chain FAMEs that are often 368 more stable than short chain FAMEs and may be more resistant to diagenetic processes and 369 preserved longer after entombment [START_REF] Wilhelm | Xeropreservation of functionalized lipid biomarkers in hyperarid soils in the Atacama 1091 Desert[END_REF]. Long chain FAMEs were identified in 370 nearly all surface and subsurface samples. At Hveravellir, they are present in greater abundance at 371 the mid-and distal apron sites than the vent site. At Gunnuhver, long chain FAMEs are also present 372 in the mid-and distal-apron sites, and present at a lower abundance with a noticeable absence in 373 the vent sites. This increase in abundance is consistent with models of lower temperatures (<40°C) 374 downstream from vents, which support plant growth and the potential for their entombment in the 375 sinter [START_REF] Walter | Hot-spring sediments in Yellowstone National Park[END_REF]. At Lýsuhóll these FAMEs are present in the relict vent sites both on the 376 surface and in the subsurface. The surface FAME load in the Hveravellir spring system varied greatly between the vent, mid-390 apron, and distal apron facies. The abundance of FAMEs detected with flash pyrolysis at the opal-

391

A vent facies is an order of magnitude (>35000 ng total FAME/ mg TOC) greater than the FAME 392 abundance at the amorphous silica distal apron (ca. 1875 ng total FAME/ mg TOC). The 393 amorphous silica mid-apron FAME abundance was lower still, at ca. 600ng total FAME/ mg TOC.

394

The elevated FAME abundance at the Hveravellir vent sample indicates that the change in water Castenholz, 1969) or perhaps diatoms (e.g. Figure 4), or both.

404

This is consistent with the relatively low abundance and lack of C 18:ω9 FA in the subsurface sample.

405

As we report on and discuss our results for the sinter samples below it is important to keep in mind 406 that these hydrothermal siliceous springs are dynamic systems suited for the production and indicates that lower thermal tolerance bacteria have recolonized this recently ceased vent (i.e. no 410 water flow). The expected trend for an active high-temperature spring system would include a 411 higher fatty acid abundance further from the high temperature vent (Kaur et al., 2011), as a greater 412 diversity of organisms with higher growth rates tend to colonize these regions.

413

The opal-A pisolith layer was collected further away from the other three Hveravellir samples but et al., 1978;Folk and Lynch, 2001;419 O'Reilly et al., 2017). Therefore, the trends in presence and preservation of FAMEs in the pisolith 420 layer is expected.

421

The FAME load at Hveravellir changed with depth between the vent, mid-apron, and distal apron 422 facies as well. Most subsurface FAMEs decreased in abundance or were not detected relative to 423 their complementary surface samples (e.g. Figure 8, where the total organic carbon load is 424 generally lower in subsurface samples relative to surface samples). A few exceptions exist in this 425 data set, in particular in the mid-apron sample, in which all of the subsurface FAMEs were elevated 426 in concentration relative to the surface (Figure 5). In the distal-apron samples, several individual 427 subsurface FAMEs were elevated relative to their complementary surface samples. This trend was 428 distinct at the distal apron site, where FAME concentration often increased stepwise from the 429 surface to progressively deeper sampling depths (e.g. Figure 5). We interpret this trend to represent 430 either a temporary increase in organic preservation potential at spring sites that experience lower The FAME load in this system changed with depth more dramatically than in the Hveravellir [START_REF] Shiea | Comparative analysis of extractable lipids in hot 1037 spring microbial mats and their component photosynthetic bacteria[END_REF]Pancost et al., 2005Pancost et al., , 2006;;Zhang et al., 2007, Kaur et al., 2011). Often branched or 540 unsaturated fatty acids are prevalent in younger deposits (Kaur et al., 2011), as was also 541 demonstrated in the work presented here. In an actively flowing spring system, temperature in 542 particular tends to drive strongly influence the length and therefore abundance of fatty 543 acidshydrocarbons, such as bacterial diethers in active geothermal systems (Kaur et al., 2015).

544

Thermophilic organisms tend to have longer higher molecular weight diethers with higher 545 thermostabilities, longer fatty acid chains (Oshima and Miyagawa, 1974;Russell, 1984; 546 [START_REF] Weerkamp | Effect of temperature on the fatty acid composition of the 1082 extreme thermophiles, Bacillus caldolyticus and Bacillus caldotenax[END_REF], increased the ratios of iso-to anteiso-branched fatty acids (Oshima 547 and Miyagawa, 1974;[START_REF] Shen | Fatty Acid Distribution in Mesophilic and 1034 Thermophilic Strains of the Genus <em>Bacillus</em&gt[END_REF], and fewer unsaturations and branches (Daron, 1970;Ray 548 et al., 1971). All of these adaptations serve to maintain optimal membrane fluidity at higher 549 temperatures.

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Although the Hveravellir vent sampled here was no longer actively flowing, several of these trends 551 were observed (Figure 9), consistent with the recent cessation of flowing spring water. and the iso to anteiso ratio trend was consistent at this site between the surface and subsurface 561 samples. Additionally, the ratio of branched to total fatty acids increases with distance from the 562 higher temperature vent, consistent with a decrease in the dominance of any thermophilic 563 organisms.

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In contrast, the inactive Gunnuhver site had a relatively consistent, elevated total FAME is within the range of the Hveravellir spring site, suggesting that these Lýsuhóll vents were higher 575 temperature when flowing and the thermophile FAMEs are well preserved. The iso to anteiso ratio 576 was lower than at the modern inactive spring sites, suggesting degradation of the branched fatty 577 acids from an original, higher temperature population. The ratio of branched to total fatty acids, 578 however, was comparable to or higher than the Hveravellir vent site, indicating that more branched 579 fatty acids relative to the total fatty acid load are preserved. This trend may be due to a greater 580 initial branched fatty acid load that, along with the total fatty acids, has experienced degradation.

581

The branched to total fatty acid ratio at Vent #3 (160807.12) is higher than all other samples and 582 contains the longest ACL of all samples from this study. This vent is composed solely of travertine, 583 which may introduce a preservation bias between this younger calcite deposit and the other vents 584 that are older and silica-dominated. The high branched to total fatty acid ratio and ACL value of 585 Vent #3 may also correspond to an active mesophilic microbial community at this site. process, and specifically the transition to microquartz, reduces porosity and decreases the 621 preservation of organic matter in siliceous sinter [START_REF] Teece | Biomolecules from fossilized hot-spring sinters: implications for the search for life on Mars[END_REF].

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These trends in mineralogy and organics preservation were upheld in the current study. In the

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Hveravellir system, which is composed of amorphous silica and opal-A, the diversity and 624 abundance of FAMEs was elevated in both the surface and subsurface. In contrast, the inactive >80 °C up to a maximum of 110 °C where elevated hydrostatic pressure allows waters to reach 669 higher temperature without boiling [START_REF] Segerer | Life in 1032 hot springs and hydrothermal vents[END_REF]. For reference, the upper temperature 670 limit for archaeal life is 121 °C in submarine black smoker environments (Kashefi and Lovley, 671 2003); in contrast, photosynthetic microbes are present in waters below 73 °C [START_REF] Ward | Cyanobacteria in Geothermal Habitats[END_REF].

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Although these organisms do not directly contribute to silica precipitation in these spring systems, 673 they can be entombed and preserved in the rapidly precipitating silica, contributing to the organic 674 load and microstructures found in near-vent environments (Cady and Farmer, 1996). In our study, 675 silicified traces of microorganisms are readily identified in SEM images from the Hveravellir and 676

Lýsuhóll sample sites, but no distinct biogenic features were identified from the Gunnuhver sinter 677 samples (Figure 4). These observations appear to correlate with the relative abundances of organics 678 detected at these three locations. 2015)). These oxidation processes are expected to occur both on the surface and in the subsurface of Mars.

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Ionizing radiation is another serious concern for the long-term preservation of Martian organics.

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The ionizing radiation dose on the surface of Mars is estimated at 0.54-0.85 Gray/year (Dartnell

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The Curiosity rover can drill to a maximum of six centimeters and the Rosalind Franklin rover . Fatty acid distribution across hot spring facies, with locations "vent", "mid-apron" and "distalapron" representing environmental facies as determined by textural characterization. A) Total ng of FAMEs / g TOC. The total FAME concentration at the Hveravellir site is highest at the vent, whereas the concentration at the Gunnuhver site is highest at the distal apron. B) The average carbon chain length at the Hveravellir site slightly increases from vent to distal apron, whereas at the Gunnuhver site the average chain length dramatically decreases. C) The ratio of iso-to anteiso-C15 is highest in the Hveravellir vent site and lowest in the distal apron facies. No br-FA were detected in the Gunnuhver site. The br-FA ratio was low for the Lýsuhóll vent sites. D) The ratio of branched fatty acids to total fatty acids was lowest at the Hveravellir vent site and highest at the distal apron site. No br-FA were detected in the Gunnuhver site. The ratio of br-FA to total FA was higher for the Lýsuhóll vent sites. x X x X x X x X X x X x X x X x x x X IC160730.09.I

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  Different wet chemistry reagents can be used based on the chemistry of the targeted labile group ;(Goesmann et al., 2017; Mahaffy et al., 2012), N,N-123 dimethylformamide dimethyl acetal (DMF-DMA; Goesmann et al., 2017), trimethylsulfonium 124 hydroxide (TMSH; Ishida et al., 1999), and tetramethylammonium hydroxide (TMAH; Grasset et 125 al., 2002; Mahaffy et al., 2012), to name just a few. Thermochemolysis with TMAH in methanol 126 (MeOH, a solvent) allows access to refractory or insoluble organic material, including 127 macromolecular organics such as kerogen-like compounds. Simply pyrolyzing a sample at high 128 temperature (>650°C) results in fragmentation of the parent molecule due to thermal stress, which 129 limits molecule identification. To cleave these refractory molecules at a milder temperature (e.g. 130 ≤600°C

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  not substantially affect the recovery of fatty acids liberated with TMAH thermochemolysis (He et 142 al., submitted), making TMAH an ideal reagent to use in the search for select organics on Mars. 143 Although the SAM instrument TMAH experiment on the Curiosity rover has yet to be performed 144 (as of the time of this writing), several other lines of evidence already indicate that organics are 145 present on the surface and near-surface (<6 cm) of Mars. Chlorinated hydrocarbons such as 146 chlorobenzene and C 2 to C 4 dichloroalkanes were detected in the Sheepbed mudstone in

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  identified in exogenous carbonaceous material, such as in the Murchison meteorite (Cronin et al., 162 1993). Biotic and abiotic-generated carboxylic acids produce distinctly different profiles in GC-163 MS analyses. Carboxylic and fatty acids derived from metabolic processes can range from C 2 to 164

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  specifically fatty acids, in modern inactive and relict Icelandic hydrothermal spring systems that 173 precipitate siliceous sinter and travertine. Organics preservation in these Mars-analog hot spring 174 environments is compared between mostly inactive and relict spring deposits and include samples 175 from spring vent, mid-apron and distal apron facies, and within a depth range comparable to the 176 sampling depth of the Curiosity and future rover drill systems. We present data to assess the limits flight-like analyses and how returned in situ data from extraterrestrial hot springs may be at three Icelandic hydrothermal sinter sites, at the surface and subsurface 182 ranging from 2 to 18 cm in depth from spring vent to distal apron, to explore the coupling of system 183 activity with potential preservation of biosignatures. In hydrothermal hot-spring systems there are 184 three main regions: near-vent, the location of higher-temperature fluid discharge from the 185 subsurface; the middle-apron, a moderate temperature region often with pooling water; and the 186 distal-apron, areas of cooler fluid further from the vent (e.g. Figure1). A currently active region, 187 Hveravellir, sits in the central highlands area of Iceland (Figure3B) and contains many hot springs 188 and fumaroles. This area is protected as a monument in Iceland, with boardwalks and restricted 189 sites that preserve the formations and minimize human alteration. At Hveravellir, we collected 190 sample 160730.01 (mid-apron) near an active spring vent, but sampling site was only moist and 191 not within the main flow region (Figure4). We also sampled at a recently inactive vent (160730.09) 192 and the distal-apron (160730.08) of an inactive vent. The pisolith sample (160730.04) had formed 193 in pools at an actively flowing vent. Given the variety of samples from active and recently inactive 194 sites, the Hveravellir system will be referred to as 'inactive' in this work. The geothermally active 195 Hveravellir region has been geochemically characterized, with the spring vents in the area 196 generally emerging at ca. 90 °C, with a pH of 8.60 and [SiO 2 ] of 599 ppm (Camacho, 2017). The 197 actively flowing spring vents proximal to Hveravellir vent sample "160730.09" ranged from 91.0 198 to 97.1°C, pH 8.15 to 8.65, [SiO 2 ] of 321 to 641 ppm, and [Cl] of 53.1 to 63.6 ppm. Hveravellir 199 spring waters are considered part of a population of high-temperature geothermal waters, along 200 with Geysir, Hveragerdi, and Reykjadalir, which contain elevated silica concentrations relative to 201 the lower-temperature geothermal regions on the island.

  thermal springs are present nearby in the Snӕfellsnes region(Camacho, 2017). Silica sinter 211 (e.g. Opal-A) deposits constitute the mineralogy of older regions in this deposit, overprinted by 212 younger travertine deposits(Jones and Renaut, 2017, Jones et al., 2005). Vent and near-vent 213 samples were collected at Lýsuhóll. The determination of vent locations was more difficult at this 214 relict site as erosion had disrupted many of the deposits; therefore only three well preserved sites 215

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  were permitted to sample the Gunnuhver site by the National Energy Authority. Lýsuhóll is private 219 land for which we had owner permission to sample. Lastly, we also had permission from the 220 Icelandic Institute of Natural History to transport samples out of Iceland. , all sinter samples were collected using solvent-washed and ashed 223 tools, while wearing gloves, facemasks and hairnets. Samples were collected using solvent cleaned 224 (MeOH -MeOH/DCM -DCM) and ashed (at 500°C) chisels, spoons, and tweezers. Rock 225 hammers were covered in a new piece of ashed (at 500°C) foil before use. Samples were placed in 226 solvent washed and ashed (at 500°C) glass jars and stored on ice for transport back to the 227 laboratory. Samples were collected both from the surface (denoted with an 'S') or from several 228 centimeters into the subsurface (denoted with an 'I'). Fresh solvent-washed tools were used for 229 each surface and subsurface sample to limit cross-contamination between sample depths. Samples 230 were transported to the laboratory on ice and stored in a -20 °C freezer upon arrival. Samples for 231 SEM analysis were either collected in the field in sterile whirlpak bags with solvent washed tools, 232

395

  flow and/or temperature at this recently ceased vent has likely led to colonization by bacteria with 396 a lower temperature tolerance. Water flow and temperature are major factors in determining 397 microbial diversity and biofilm texture in hydrothermal systems. When flow shifts, as it has at this 398 vent and outflow system, change to abundance and diversity of the fatty acids should be expected. 399 At the vent, the presence of the C 20 and C 22 fatty acids indicate that thermophilic bacterial growth 400 has occurred, however, the relatively high FA abundance, together with an increase in the 401 cyanobacterial C 18:ω9 indicates that a reduction in water temperature probably resulted in increased 402 growth of a thermotolerant cyanobacterium such as the high-temperature form of Mastigocladus 403 isolated from an Iceland hot spring (

414

  exhibits a similar trend in even-over-odd carbon number predominance, with n-C 16:0 and n-C 18:0 415 dominating the FAME profile. Pisoliths are composed of >2 mm diameter spherical concretionary 416 grains called pisolites which are often formed from calcite or amorphous silica. Due to the 417 concretionary nature of the grains, pisolites and related ooids often contain bound organic matter 418 between cortices or submillimeter concretion layers (Davies

431

  temperatures and diagenetic alteration, or a modern and robust subsurface microbial community 432 that is generating the elevated FAME abundance. This trend could also be an indication of 433 preservational bias related to differences in mineralization rates, where early mineralization 434 minimizes the degradation of organics and body fossils(Alleon et al., 2016). If a diversion of water 435 flow precipitated a localized temperature shift, the supply of dissolved silica would also decrease 436 leaving the microbial community more vulnerable to biotic and abiotic degradation processes since 437 it was not mineralizing. These observations reinforce the complexity of these spring systems.

  471system. Subsurface FAME abundance decreases substantially or to no detection relative to their 472 complementary surface samples, indicating continued degradation of the FAMEs in the subsurface 473 due to diagenesis and microbial recycling. Only four FAMEs (n-C 8:0 , n-C 10:0 , n-C 15:0 and trans-474 C 18:1 ) are detected in the subsurface distal apron site with flash pyrolysis(n-C 8:0 , n-C 10:0 , n-C 15:0 475 and trans-C 18:1 ).

565

  concentration at the vent and mid-apron facies, and a much higher FAME concentration at the 566 distal apron facies, consistent with secondary recolonization of the sinter by more diverse, lower 567 temperature mesophiles after spring flow ceased. Average chain length also decreases from the 568 vent facies (ACL = 10.7 to 13.4 at vents) to the distal apron facies (ACL = 0 to 8.9), suggesting 569 the degradation of fatty acids to shorter chains due to heterotrophy or diagenesis.570Only relict inactive vents were sampled at the Lýsuhóll spring site, which allows a comparison 571 with the modern inactive vent systems. The total FAME concentrations at the relict site was within 572 the range of the inactive spring sites, with higher abundances in the surface samples and lower 573 abundances in the subsurface samples. Average chain length at the relict site (ACL = 14.9 to 17.4) 574

724

  et al., 2007). Ionizing radiation destroys biomolecules (e.g. proteins or DNA), and most bacteria 725 cannot survive doses greater than 200 Gray (Daly, 2009). Recent studies indicate that within 300 726 million years, preservation of functionalized organic molecules greater than 500 amu in the top ~5 727 cm of rock drops 1000x due to ionizing radiation (Pavlov et al., 2012). There is some indication 728 that rapid burial of organics, short exposure ages due to relatively slow Martian weathering rates, 729 and the characteristics of some entombing mineralogies may assist in organics preservation in the 730 Martian near-surface (Hays et al., 2017 and references therein), but the need remains to sample the 731 Martian subsurface to reduce the impact of degradation via radiation effects.

733

  will be able to drill to two meters depth. The work presented here indicates that even with 734 diagenetic effects and heterotrophic cycling of organics, organic molecules are preserved and 735 detectable over short timescales in the near-subsurface (2 to 18 cm in depth) of modern and relict 736 terrestrial hot springs. Organics therefore may be detectable on Mars in relatively recent hot spring 737 deposits, but preservation likely deteriorates over geologic time scales. These depths are within 738 the drill sampling range of the Curiosity and Rosalind Franklin rovers (partial to complete depths, host unique microbial ecosystems and have the capacity to 743 physically preserve both microbial communities and their associated organic matter as 744 biosignatures within siliceous sinter. These terrestrial geothermal springs serve as Mars analog 745 environments to prepare future Mars missions to target their search for biosignatures in Martian 746 hot spring deposits. This study quantified the preservation and detectability of fatty acids in three 747 Icelandic hot spring deposits ranging from modern and inactive to relict, and samples were 748 collected both at the surface and at depth (ranging from 2 to 18 cm in depth). Analyses performed 749 with TMAH thermochemolysis pyrolysis-GC-MS indicate that fatty acids were preserved and 750 detectable in all three spring deposits, with the subsurface samples degraded relative to the surface 751 samples. TMAH thermochemolysis pyrolysis-GC-MS analyses using a SAM-instrument-like 752 procedure were also successful in liberating preserved fatty acids from these sinter samples, albeit 753 generally in lower abundances and with less diversity. These results build confidence in our ability 754 to detect fatty acids and distinguish biogenicity using the GC-MS instrumentation available on 755 current and future Mars missions. sample collection was provided by the NASA Planetary Science and 758 Technology through Analog Research (PSTAR) program grant #NNX15AJ38G to J.R. Skok and 759 the American Philosophical Society's Lewis and Clark Fund for Exploration and Field Research 760 in Astrobiology to K. Craft. S.S. Johnson would like to acknowledge support from NASA grant 761 #NNX17AJ68G ("Using Organic Molecule Detections in Mars Analog Environments to Interpret 762 the Results of the SAM Investigation on the Mars Science Laboratory Mission") as well as a

F

Figure 3 .

 3 Figure 3. A) Map of field locations in Iceland, with the capitol of Reykjavík for reference. B-D) Field site numbers at the Hveravellir, Gunnuhver, and Lýsuhóll spring systems. Stars at Hveravellir indicate active flowing spring vents where geochemical measurements were taken. 209x180mm (150 x 150 DPI)

Figure 4 .

 4 Figure 4. Field sample site images and corresponding SEM images of sinter from A-C) Hveravellir (active vent in top left of panel A), D-F) Gunnuhver, and G-I) Lýsuhóll. White box indicates zoom image B, E, or H. Rock hammer for scale in surface images. C) Silificied diatoms, microbial filaments, and putative plant matter present in the Hveravellir site. F) Opal-CT accretionary spheres with no confirmed biogenic features present in the Gunnuhver site. I) Abundant silicified casts of microbial filaments present in the Lýsuhóll site. 229x179mm (150 x 150 DPI)

Figure 5 .

 5 Figure 5. FAME profile for the active Hveravellir Hot Spring system analyzed with a 500 °C flash pyrolysis step. 339x190mm (150 x 150 DPI)

Figure 9

 9 Figure9. Fatty acid distribution across hot spring facies, with locations "vent", "mid-apron" and "distalapron" representing environmental facies as determined by textural characterization. A) Total ng of FAMEs / g TOC. The total FAME concentration at the Hveravellir site is highest at the vent, whereas the concentration at the Gunnuhver site is highest at the distal apron. B) The average carbon chain length at the Hveravellir site slightly increases from vent to distal apron, whereas at the Gunnuhver site the average chain length dramatically decreases. C) The ratio of iso-to anteiso-C15 is highest in the Hveravellir vent site and lowest in the distal apron facies. No br-FA were detected in the Gunnuhver site. The br-FA ratio was low for the Lýsuhóll vent sites. D) The ratio of branched fatty acids to total fatty acids was lowest at the Hveravellir vent site and highest at the distal apron site. No br-FA were detected in the Gunnuhver site. The ratio of br-FA to total FA was higher for the Lýsuhóll vent sites.

Figure 10 .

 10 Figure 10. Comparison of FAME concentrations determined with flash pyrolysis experiments from the surface and subsurface vent sites from the Hveravellir, Gunnuhver, and Lýsuhóll spring systems. The even-over-odd carbon number preference is most apparent in the Hveravellir and Gunnuhver locations. 242x180mm (150 x 150 DPI)

F

  o r P e e r R e v i e w O n l y ; N o t f o r D i s t r i b u t i o n Group Sample ID

  y ; N o t f o r D i s t r i b u t i o n

  y ; N o t f o r D i s t r i b u t i o n

  bound within macromolecules. Wet chemistry experiments aim to transform 117 polar organic molecules (e.g. carboxylic acids) into volatile derivatives that are amenable and

	F o r F o r F o r 26 F o r F o r r o F 116 and liberates organics
	P e P e P e P e P e P e
	e e e e e e
	r r r r r r
	R R R R R R
	e v e v e v e v e v e v
	i i i i i i
	e w e w e w e w e w e w
	O n O n O n O n O n O n
	l y l y l y l y l y l y
	; ; ; ; ; ;
	N N N N N N
	o t o t o t o t o t o t
	f f f f f f
	o r o r o r o r o r o r
	D D D D D D
	i i i i i i
	s t s t s t s t s t s t
	r i r i r i r i r i r i
	b u b u b u b u b u b u
	t t t t t t
	i o i o i o i o i o i o
	n n n n n n
	2 5

118

detectable by

. FAMEs Detected with 500 °C Instantaneous Flash Pyrolysis 299

	F o r F o r 265 0.25 µm thick Crossbond® 5% diphenyl/95% dimethyl polysiloxane stationary phase, and He as F o r 294 at Gunnuhver was predominantly opal-CT, with the distal apron being dominantly amorphous 295 silica. This system also contained lesser percentages of halite and quartz. The older relict hot spring 296 at Lýsuhóll is composed almost exclusively of amorphous silica with some minor calcite, and one 323 n-C 18:0 were the most abundant FAMEs in most samples, although this is not the case for Vent #1 F 324 (160809.03). FAMEs longer than n-C 18:0 were identified at all sites. The abundance and diversity o r 325 of FAMEs was increased in the surface samples relative to the subsurface samples, which ranged
	297 326	P e P e P e sample site was composed entirely of calcite. e P from 2 to 3 cm in depth.
	270 298 327	e r e r (Agilent Technologies). Identifications were based on comparison to known FAME retention e r r 3.2Samples were initially analyzed using a 500 °C flash pyrolysis technique to enable e 3.3
	271 300	R R times of a Supelco 37 component FAME mixture that contains saturated FAMEs, monounsaturated R transesterification of fatty acids with TMAH thermochemolysis. Using this technique there was R
	272 301 302	e v i e v i fatty acid methyl esters (MUFAMEs), and polyunsaturated fatty acid methyl esters (PUFAMEs) e v i relatively high diversity and abundance of FAMEs in the Hveravellir spring site, a lower diversity e v and abundance of FAMEs at the inactive Gunnuhver spring site, and diminished diversity and i
	303 304	e w e w e w abundance of FAMEs at the Lýsuhóll relict site (Table 2). At the Hveravellir site, samples were collected from the spring vent, mid-apron, and distal-apron, e w
	280 305 306 307	O n l y ; O n l y ; O n l y as well as a pisolith-bearing layer near an active vent. FAMEs detected ranged from n-C 6:0 to n-for 2 minutes, then ramped at 80 °C/min to C 27:0 and included a few monounsaturated fatty acids (MUFAs) and branched iso-and antiso-C 15:0 O n (Figure 5, Table 2). n-C 16:0 and n-C 18:0 were the most abundant FAMEs in all samples, and FAMEs l y 305 °C and held for 3 minutes. The GC column program started at 35 °C and was held for 10.9 ; ;
	281 282 283	N o t N o t minutes, then ramped at 15 °C/min to 175 °C with no hold, then ramped at 10 °C/min to 305 °C N N and held for 1 minute. The auxiliary transfer line was held at 135 °C. For the 500 °C flash pyrolysis method, the sample was dropped into a 500 °C pyrolyzer oven and o t o t
	284 285	f o r f o r f f held for 1 minute. The inlet program and Tenax ® TA trap started at 15 °C with a 2 minute hold. The inlet was ramped at 900 °C/min to 300 °C and held for 2 minutes. The GC column program o r o r
	286 287	D i D i started at 35 °C and was held for 2.5 minutes, then ramped at 5 °C/min to 300 °C and held for 5 D D minutes. The auxiliary transfer line was held at 270 °C. i i
	288	3. Results	s t r i s t r i s t s t r i r i
	289 290	b u b u The mineralogy of the three sample locations was generally dominated by opal-A, opal-CT, and 3.1. Mineralogy b u b u
	291 292	t i o t i o t t amorphous silica (Table 1). The modern inactive hot spring vent at Hveravellir was dominantly opal-A with lesser percentages of clinopyroxene, plagioclase, gypsum, and magnesite. Several i o i o
	293	n n Hveravellir locations were composed entirely of amorphous silica. The modern inactive hot spring n n
		10 11 12

266

the carrier gas at a 3 mL min -1 flow rate and 10:1 split. Blank (without TMAH) and "TMAH-267 blank" (with TMAH) clean up analyses were run between each sample to determine and subtract 268 any background level of residual FAMEs in the column. 269 Chromatograms and the mass spectra for FAMEs were analyzed with ChemStation software 273 (Sigma-Aldrich). In addition, mass spectra were compared to the National Institute of Standards 274 and Technology (NIST) Spectral Library. Quantifications were based on integration under peaks 275 and scaled to the known amount of the n-C 19:0 internal standard.

276

The pyrolyzer and GC programs used are detailed below: For the SAM-like pyrolysis ramp and 277 GC column method, the pyrolyzer program started at 50 °C and was ramped at 35 °C/min to 400 278 °C with no hold. The inlet program and Tenax ® TA trap started at 15 °C with a 2 minute hold.

279

The inlet was ramped at 68 °C/min to 150 °C and held

. FAMEs Detected with SAM-Instrument-like Pyrolysis Ramp 328

	350 F o 4. Discussion The samples studied here comprise silica sinter and calcite hot spring deposits ranging from 351 r 352 modern and partially inactive to older, relict spring systems. Thus, they represent a range in P 353 geochemistry, mineralogy, age, and diagenetic history, as well as spanning a diversity of e 354 subenvironments present in hot spring deposits: vents, mid-aprons, and distal aprons. Despite these e 355 differences, several trends in FAME abundance and diversity are shared between the hot spring r Samples were analyzed using a 35°C/min pyrolysis ramp to approximate the pyrolysis ramp used 329 by the SAM instrument on the Curiosity rover (Mahaffy et al., 2012; Williams et al., 2019). This R 356 locations.
	330 331 357 358 332 333 359 360 361	technique was utilized to explore how the FAME detection would change if sinter-bearing hot A high abundance of n-C 16:0 and n-C 18:0 is observed in all of the surface and subsurface samples e v relative to other FAMEs in all three locations, which is consistent with these two fatty acids being springs were analyzed on Mars with a SAM comparable TMAH experiment. In general, with this technique there was lower diversity and abundance of FAMEs in all three spring systems (Table 3). i the most common in both bacteria and eukarya (Kaur et al., 2015; Johnson et al., 2019; Wilhelm e et al., 2017; Williams et al., 2019). Elevated n-C 16:0 and n-C 18:0 FAME abundances relative to other w FAMEs are expected in younger sinters versus much older, inactive sinters (Kaur et al., 2011),
	334 335 336 337 362	At the Hveravellir site, FAMEs detected ranged from n-C 6:0 to n-C 20:0 and included two MUFAs. n-C 16:0 and n-C 18:0 were the most abundant FAMEs in all samples, and only one FAME longer of FAMEs was equal to or increased in the surface samples relative to the subsurface samples. y l than n-C 18:0 was identified at the surface mid-apron site. In general, the abundance and diversity n O
		;
	338 339 340	At the inactive Gunnuhver site, FAMEs detected ranged from n-C 4:0 to n-C 2718:0 and included N severalone MUFAs and PUFAs. n-C 16:0 and n-C 18:0 were generally the most abundant FAMEs in the mid-and distal-apron samples, and no FAMEs longer than n-C 18:0 were detected in all o t
	341	Gunnuhver samples. No FAMEs larger than n-C 16:0 were detected in the Vent 2 site. The f
	342 343	abundance and diversity of FAMEs was uniformly higher in the surface vent samples relative to the subsurface samples, but lower than the subsurface samples from the mid-and distal apron o r
	344	environments, with no FAMEs detected in the subsurface Vent 2 sample. D
		i
	345 346 347 348 349	At the relict Lýsuhóll site, FAMEs detected ranged from n-C 4:0 to n-C 18:0 and included three s t MUFAs and one PUFA. n-C 16:0 and n-C 18:0 were the most abundant FAMEs in all samples, and no r i FAMEs longer than n-C 18:0 were detected. The only site with both a surface and subsurface sample was the 160807.04 Vent #2 location, and the abundance and diversity of FAMEs was higher in the b u surface sample relative to the subsurface sample. t
		i o
		n
		13

Organics Load in the Hveravellir Hot Spring System 389

  Much higher concentrations of long chain FAMEs in the surface

	F o r
	P e
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	r
	R
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377

samples suggest a more modern source. Observation of the long chain FAMEs at depth suggests 378 that plant matter grew and was entrained in the sinter as flow decreased and the spring system 379 cooled and became dormant. The lower concentrations in the subsurface samples indicate some 14 380 higher plants were also incorporated into the rock record, with some degradation of the FAMEs 381 due to burial processes.

382

A strong even-over-odd carbon number preference is consistent with the production of the fatty 383 acids due to enzymatic effects (e.g. the monomer addition of a 2-C sub-unit,

(Georgiou and 384 Deamer, 2014;[START_REF] Summons | Molecular Biosignatures[END_REF]

and of microbial cellular metabolic processes

(Elias et al., 385 1997; Grimalt and Albaiges, 1987; Nishimura and Baker, 1986;[START_REF] Volkman | Lipid biomarkers for marine organic matter[END_REF]

. All surface and 386 subsurface samples from the Hveravellir site exhibit this trend, as do all surface samples from 387 Gunnuhver. 388 4.1.

2. Organics Load in the Gunnuhver Hot Spring System

  FAMEs are not less well preserved further from the vent and are likely degraded to shorter chains,

	F o r 439 water flow) can have cascading impacts on the microbial composition and their ecology, that 440 together end up biasing their fossil record. r o F 468
	442 443 444	P e e r contamination, we cannot rule out the possibility that some of these samples reflect an P e anthropogenic influence, as this hot spring is a tourist destination and the mid-apron sample was e collected below a visitor boardwalk. Elevated FAMEs from eukaryotic (e.g. human) sources may r
	445	R have been entrained in the sinter. Contamination of this type should yield a much higher FAME R
	446 447	e v concentration, especially in the C 16:0 and C 18:0 FAMEs, in the surface relative to the subsurface samples. Surface samples did contain higher concentrations of FAMEs than the subsurface, but e v
		i i
		e w e w
		O n O n
		l y l y
		; ;
		N N
		o t o t
		f f
		o r o r
		D D
		i i
		s t s t
	463	r i elevated in the amorphous silica distal apron facies (ca. 37000 ng total FAME/ mg TOC). This r i
	464 465	b u trend in abundance is expected, as a greater diversity of organisms with higher growth rates tend to colonize lower temperature geothermal regions such as the distal apron. b u
		t t
		i o i o
		n n
		16 17

438

While general trends can be preserved, changes in the local physical conditions, (like diverted 441 Although our sampling techniques were optimized to decrease any chance of anthropogenic 448 C 16:0 and C 18:0 FAMEs were not uniformly elevated. Therefore, we interpret the Hveravellir FAME 449 concentration trends as reflecting a diverse and active microbial community.

450

In general, branched and unsaturated fatty acids were either not detected in subsurface samples or 451 were decreased in abundance relative to the surface samples. This degradation was likely as the 452 unsaturation in the fatty acid is a more reactive location for the molecule to be cleaved by microbial 453 heterotrophy or diagenesis (Killops and Killops, 2005; Sun et al., 1997). A decrease in unsaturated 454 fatty acids and branching is documented in older sinter samples (Kaur et al., 2011) and is consistent 455 with the expectation for preferential degradation of unsaturated fatty acids. However, this trend 456 was reverse in the mid-apron sample, in which all of the subsurface FAMEs, including branched 457 and MUFAs were elevated in concentration relative to the surface.

458 4.

459

The surface FAME load in the inactive Gunnuhver spring system also varied between the vent, 460 mid-apron, and distal apron facies, although to a lesser degree than in the Hveravellir spring 461 system. The abundance of FAMEs detected with flash pyrolysis is comparable between the opal-

462

CT vent and opal-CT mid-apron facies (ca. 18,000 to 20,000 ng total FAME/ mg TOC) and is

466

The abundance and diversity of lower molecular weight FAMEs increases to the mid-apron and 467 distal apron facies relative to the vent facies, suggesting that higher molecular weight (longer) 469 potentially due to microbial heterotrophy. 470

Comparison between Flash Pyrolysis and Flight-like Ramp Pyrolysis

  The subsurface FAME profiles exhibit strong to weak even-over-odd trends in 476 carbon number. The subsurface distal apron sample does not exhibit any trends in carbon number Vent #2) surface sample to ca. 1500 ng total FAME / mg TOC in the 499 complementary subsurface sample. This indicates continued degradation of the FAMEs in the 500 subsurface due to diagenesis and microbial recycling. The Lýsuhóll samples exhibit a moderate to Correlations vary between the number of FAMEs detected, the concentration of FAMEs in the 522 sample suite. In the Hveravellir system, the greatest diversity of FAMEs detected was generally as were some of the highest % TOC values. In particular, n-C 16:0 concentrations 529 were predictably low in the subsurface samples and high in the surface samples. These lines of 530 evidence indicate the relict system is experiencing higher delivery of organic load now than it 531 perhaps did in the past. The organics that constitute these higher % TOC values may represent a

	F o r P e e r R e v 498 in the Vent 160807.04 (501 weak even-over-odd preference, which may indicate degradation of fatty acids from the original 502 microbial community, and limited input of organics from a modern community. The relict 503 Lýsuhóll system is the only site where the PUFA trans-C 18:2 algal biomarker is detected (Campbell 504 505 506 preferential degradation of these fatty acids. v e already limited in detection at the surface, a dearth that continues into the subsurface and indicates R et al., 2015), and then only in two subsurface samples. Branched and unsaturated fatty acids are F o r P e 528 far greater diversity of organic molecules than just fatty acids, which are preserved but in low spring system, 532 e r 533 concentrations in the relict system.
		i i
		e w e w
		O n O n
		l y l y
		; ;
		N N
		o t o t
		f f
		o r o r
		D D
		i i
	492 493 494 495 523 524	s t s t the most readily identified in older, degraded hot spring deposits. The surface FAME load in the r i r i relict Lýsuhóll system also varied between the vent facies, ranging from ca. 3,700 ng total FAME/ mg TOC in the amorphous silica and calcite dominated Vent #2, to ca. 350 ng total FAME/ mg b u correlated with low % TOC. This diversity is invaluable in assessing microbial community composition, even at low concentrations. In the inactive spring system, the greatest diversity of b u TOC in the amorphous silica Vent #1, to ca. 83 ng total FAME/ mg TOC in the calcite Vent #3 t t
		i o i o
		n n
		18 19

477

or molecular weight, which likely reflect whole-scale degradation of all FAMEs in the subsurface. 478 Mono-and polyunsaturated fatty acids are already limited in detection at the surface, a dearth that 479 continues into the subsurface and indicates preferential degradation of the unsaturated FAMEs. 480 Branched fatty acids are not detected in neither the surface nor subsurface samples with flash 481 pyrolysis.

482

4.3. Organics Load in the Lýsuhóll Hot Spring System

483

In contrast with the opal and amorphous silica-dominated Hveravellir and Gunnuhver sites, the 484 Lýsuhóll relict spring system is composed of both amorphous silica and calcite, with Vent #3 485 composed entirely of calcite. The spring waters that precipitate these deposits, and the subsequent 486 microbial communities that colonize the springs, may be different from those present in the 487 siliceous sinter deposits

[START_REF] Zhang | Lipid biomarkers and carbon-isotopes of modern travertine deposits 1101 (Yellowstone National Park, USA): Implications for biogeochemical dynamics in hot-spring 1102 systems[END_REF]

. Additionally, several samples from the relict Lýsuhóll 488 system were destroyed and/or contaminated during transport back to the United States, reducing 489 the completeness of the surface versus subsurface samples originally collected at these sites.

490

Regardless, several conclusions can be drawn from the remaining samples about the organics load 491 in this relict system. All sample sites at Lýsuhóll represented near-vent environments as these were 496 (Table

2

). As in the other spring systems, the surface FAME concentrations are higher than in 497 subsurface samples. The total FAME abundance ranges from ca. 3700 ng total FAME/ mg TOC 507 4.4.

508

The 500 °C flash pyrolysis method, combined with TMAH thermochemolysis, represents a 509 laboratory-optimum method to release organics bound within macromolecules and sorbed to 510 minerals. The SAM instrument on the Curiosity rover, while able to perform similar experiments, 511 is limited to a 35°C/ min pyrolysis ramp. To assess how this method variation might affect the 512 detection of FAMEs in these Mars-analog siliceous sinters, samples were analyzed with the SAM-513 like 35°C/ min pyrolysis ramp.

514

The flash pyrolysis method improved FAME detection uniformly across the sample suite (Figure

515

8), and with few one exceptions, FAMEs >n-C 18:0 were only detected with the flash method. The 516 ramp method released between 30 and 86% of the FAMEs detected in the flash method. Two 517 outliers to this data set include sample 160727.03.I (200% improvement in the ramp versus flash 518 method) and 160727.02.I (no fatty acids liberated using the ramp method). Although the flash 519 method is more efficient at FAME release, it is promising for the search for organics on Mars that 520 FAMEs are also detectable with the SAM instrument pyrolysis ramp rate.

521 525 FAMEs correlates with higher % TOC, which represents an anticipated trend with regards to 526 FAME preservation (and degradation), FAME diversity and concentration, and the total organic 527 carbon load in the sample (Table

1

). The greatest diversity of FAMES was detected in the relict 534 4.5.

Mineralogic Evolution and the Preservation of Organic Matter 535

  It is important to note that the primary controls on lipid preservation in siliceous sinter deposits 536 are thermal alteration, high temperatures, and the pH of each spring(Kaur et al., 2015;[START_REF] Gibson | Bacteriohopanepolyols 864 preserved in silica sinters from Champagne Pool (New Zealand) indicate a declining 865 temperature gradient over the lifetime of the vent[END_REF]

537 al., 2006), which can vary greatly between spring systems. A great variety of lipid biomarkers have 538 been identified in modern to ca. 900 year old geothermal environments (Jahnke et al., 2001; Shiea 539 et al.

  The average 552 chain length ranges from 15.1 to 16.4 for all surface and subsurface Hveravellir samples, with the 553 exception of the surface vent sample with an ACL of 13.6. These values suggest that the surface 554 vent microbial community and/or FAME preservation is different from the other surface and 555 subsurface samples at Hveravellir. The low ACL value of the surface vent suggests that this spring the cessation of water activity that hosted the thermophiles. The ratio of 559 i-to a-C 15 is elevated near the vent where elevated temperatures affect community composition

	F o r 558 occurred concurrent with 560
	P e
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	20

556

vent never experienced very high fluid temperatures and even if thermophilic organisms were 557 present, their organic biosignatures are not well preserved. An increase in heterotrophy may have

  The vents from the three systems studied here are compared to assess the preservation of 591 organics as a concentration of FAMEs in the surface and subsurface (Figure10). Additional work

	F o r 589 diagnostic of more specific temperature and pH conditions of each subenvironment at each spring r o F 620
	592 593 594 595 596 597 598	P e e r R e v i can be completed to assess the preserved lipids separate from modern lipids in all three systems, including solvent washing sinter samples to remove surface lipids prior to analysis. At the recently P e e inactive Hveravellir vent (sample 09), the C 16 and C 18 FAMEs are likely elevated due to these being r the dominant FAMEs in both bacteria and eukarya, although anthropogenic influences, especially R in the surface sample, cannot be completely dismissed. The lower concentrations of the other FAMEs in the surface and subsurface reflect a nominal FAME profile from an active microbial e v community, with the expected even-over-odd carbon number preference. The inactive Gunnuhver i
		e w e w
		O n O n
		l y l y
		; ;
		N N
		o t o t
		f f
		o r o r
	612 613	D i s t D i although progressive silicification and preservation biases over long time scales can also destroy these physical biosignatures (Jones et al., 1997; Jones and Renaut, 1996). As siliceous sinter s t
	614	r i deposits experience diagenesis, opal-A incrementally recrystallizes to Opal-C (containing r i
	586 615 616	b u disordered α-cristobalite) or opal-CT (containing cristobalite and tridymite). This transition may occur on the order of ca. 50 years up to tens of thousands of years. Over longer time scales (ca. b u Low temperature degradative processes such as heterotrophy and diagenesis control the fatty acid t t
		i o i o
		n n
		21 22

587

distribution observed in this study. The primary fatty acid distribution from thermophilic versus 588 mesophilic communities is discernable in some environments (e.g. Hveravellir vent) but not 590 system. 599 vent generally had the lowest FAME concentrations relative to the Hveravellir and Lýsuhóll 600 systems, but still exhibited the even-over-odd carbon number preference characteristic of an active 601 microbial community. The low FAME concentrations reflects FAME degradation leading to low 602 preservation potential, and a lack of extensive recolonization of the sinter by secondary microbial 603 communities. The higher FAME concentrations at the relict Lýsuhóll Vent #2 (especially relative 604 to the inactive Gunnuhver site) likely reflect microbial (re)colonization by a community distinct 605 from that found in active/recent silica-precipitating hot springs. The mixed amorphous silica and 606 calcite mineralogy of this vent likely also contributes to this marked difference in FAME 607 abundance. 608 The mineralogic evolution and diagenesis of siliceous hot spring sinters is well studied (Herdianita 609 et al., 2000). Initial silica precipitates are composed of amorphous silica or opal-A (Tobler et al., 610 2017), and the very fine size of these precipitates are documented to preserve microbial cells in 611 extraordinary physical detail

(Cady and Farmer, 1996; Jones et al., 2000; Renaut et al., 1998)

, 617 10,000 to 50,000 years), opal-C/ -CT will reorder to microcrystalline quartz. Although sinter age 618 can sometimes be used as an estimate for crystallinity, variables such as fluid mobilization and 619 composition can also greatly affect crystallinity

(Herdianita et al., 2000)

. This recrystallization

Biomarker Preservation and Implications for Organics Detection on Mars

  then vent facies. Based solely on aerial extent, Martian hot spring apron facies 651 may be most easily detected from orbital imaging and targeted for in situ exploration.

	625 F o Gunnuhver system, which is composed primarily of opal-CT, had a much lower diversity and r 650 channel facies and 652 Biosignature preservation within all facies is complicated by preservation bias and diagenesis over P 653 geologic time scales. Even the proximal slopes and aerially extensive mid-and distal-aprons may e 654 become challenging to identify as the fine scale micro-textures that define these spring sub-e r 655 environments can be lost to diagenesis and preservation biases (Cady and Farmer, 1996). Although
	626 656	abundance of preserved FAMEs. Lynne et al. (2006) suggest that silica diagenesis is accelerated R diagenesis can and does obscure some features, the apron facies contain structures such as pools
	627 628 657 658	in hot spring systems that have transformed from flowing water to steam dominated. The transformation to opal-CT in this relatively young system was likely controlled by fluid and terracettes that are identifiable in surface deposits and layered deposits with palisade fabric e v identifiable in outcrop cross-section. Vents are just as prone to erosion and infilling (this was
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	643 644 645 646 647	flow in channel facies produces bacterial streamer fabrics, long filaments parallel to water s t direction, sometimes composed of cyanobacterial filaments (Parenteau et al., 2014). This streamer r i morphology is readily identified in outcrop (e.g., Parenteau et al, 2014, Walter et al., 1998). The lower temperatures of the distal apron are usually accompanied by thick microbial mats creating b u palisade fabrics with abundant microfossils and organics that can be preserved (Cady and Farmer, t
		i o
		n
		23

629

composition and fluid or steam mobilization. This mobilization led to accelerated diagenesis and 630 fumarolic degradation of organics in the Gunnuhver system. The surprisingly elevated diversity 631 and abundance of FAMEs in the relict Lýsuhóll system was controlled in part by the mineralogy, 632 which is dominated by calcite and amorphous silica, as well as a modern microbial community 633 that had colonized the surface of this deposit. These results further reinforce the importance of 634 ascertaining sample mineralogy prior to sampling for organics in hydrothermal systems, especially 635 on life detection missions beyond Earth.

636 4.6.

637

The size and distribution of hot spring facies varies across systems, yet trends in preservation 638 styles, from the outcrop-to the submicrometer scale, are present and can be used to provide 639 depositional context as we continue to explore life in other planets. On Earth, preservation at the 640 vent is often less favorable with microfossils usually in the form of smaller diameter molds that 641 can be readily infilled and obscured

(Cady and Farmer, 1996)

. Vents may be identified by finely 642 laminated deposits in a circular form that are often associated with geyserite. The directed water 648 1996;

[START_REF] Westall | Archean (3.33 Ga) microbe-sediment systems were 1086 diverse and flourished in a hydrothermal context[END_REF]

. Apron facies are one of the better-preserved lithofacies in degraded 649 spring systems, with the mid-to-distal apron composing the largest aerial extent, followed by the 659 observed at Lýsuhóll and Gunnuhver), and with a much smaller footprint, can be challenging to 660 identify from orbital images. However, the primary macro-scale textural characteristics of spring 661 vents can enable their identification after some geologic reworking

(Cady and Farmer, 1996, and 662

references therein).

663

Given these complexities, it is therefore important to develop a facies-oriented understanding of 664 biosignature preservation potential in terrestrial hot springs to better predict the ideal locations to 665 search for organics in Martian relict hydrothermal spring systems. The temperature of fluids at 666 subaerial hydrothermal vent systems can vary greatly, up to or above surface boiling temperatures. 667 Non-photosynthetic microbes can live in these temperatures, with hyperthermophiles preferring 668

  The search for (biogenic) organic molecules on Mars' near-surface requires new and novel 680 techniques to detect these molecules with miniaturized space-flight payloads. In the terrestrial 681 realm, many biomarkers are preserved and detectable in siliceous sinter environments with in the search for organics on Mars is the deleterious effects of oxidants and

	F o r 679 710 of organics in terrestrial systems is vital for choosing an ideal Martian hydrothermal target for F r o 711 organic biosignature exploration.
	682 683 684 685 686 687 688 713 714 715 716 717 718 719	P e e r R e v i laboratory instrumentation such as off-line lipid fraction extraction, derivatization and/or saponification, followed by multiple GC-MS and/or liquid chromatography-mass spectrometry (LC-MS) analyses. However, suites of these laboratory techniques are often too complex to be successfully miniaturized for space-flight and remote operation. Therefore, "one-step" thermochemolysis or derivatization pyrolysis GC-MS is an optimized technique that is miniaturized to work on space-flight missions such as with the SAM and MOMA instruments. To be more "SAM-like" in this study, samples that may contain low-temperature surface biofilms radiation on organics. Although organic matter has been identified in Mars near-surface substrates P e with the SAM instrument (Eigenbrode et al., 2018; Freissinet et al., 2015), fewer molecules e detected by SAM represent reduced carbon, with many of these organics being oxidized. For r example, polycyclic aromatic hydrocarbons (PAHs) and kerogen from exogenous delivery via R meteoritic infall may undergo several oxidation reactions to form stable simple organics such as e phthalic, mellitic, or oxalic acids (Benner et al., 2000; Freissinet et al., 2015) on the Martian v i surface, which are then detectable by the SAM instrument (e.g. possible source organics for
		e w e w
		O n O n
		l y l y
		; ;
		N N
		o t o t
		f f
		o r o r
	702	D i D hot spring becoming quiescent should be similar on Earth and Mars. The evolution of a putative i
	703 704 705 706 707	s t r i b u t microbial community within that hot spring can be approximated in specific ways by assessing s t microbial community evolution in hot springs on Earth. The approximations by definition r i requirerequire assumptions that do not account for Mars-specific conditions such as atmospheric composition, radiation flux, energy sources, etc. By assessing the microbial communities and b u preserved organics from lower temperature inactive and relict spring systems, this work t
		i o i o
		n n
		24 25

689

were collected and ground as whole rock samples in a manner similar to Curiosity's sample 690 acquisition system. Therefore, the results from this study likely reflect a greater abundance of 691 organics than may be expected to be preserved in a Martian hot spring deposit with a hypothetical 692 microbial community. Although more diagnostic biomolecules are present in Mars-analog 693 hydrothermal spring environments, this work demonstrates that FAMEs are preserved and 694 detectable with space-flight-like GC-MS techniques in modern inactive and relict siliceous sinter 695 and calcite deposits from Iceland. The MOMA instrument on the Rosalind Franklin rover will be 696 able to ramp the pyrolysis oven at >200 °C/min (from thermal vacuum), achieving a much faster 697 ramp than the SAM instrument and perhaps increasing the likelihood of detecting Martian FAMEs, 698 if present. 699 The rate of siliceous sinter formation, hydrothermal conditions, and those effects on biomass 700 growth and potential for silicification in each individual Icelandic sinter deposit reflect differences 701 in the diversity and abundance of biomolecules detected in this work. The geologic process of a 708 approximates the organics load in Martian hot springs with a hypothetical microbial community 709 that would have evolved as the spring system cooled to dormancy. Understanding the distribution 712 A final consideration 720 benzene in the Sheepbed mudstone, Freissinet et al. (

  J.C., Martin, F., Gonzalez-Vila, F.J., 1996. Thermally assisted hydrolysis and alkylation 823 as a novel pyrolytic approach for the structural characterization of natural biopolymers and Edgett, K.S., Watkins, J., Bridges, J.C., Maurice, S., Fisk, M.R., Johnson, J.R., Rapin, W., Des Marais, D.J.D.J., Hausrath, E.M.E.M., Horgan, B., Jahnke, L.L., Eder, W., Huber, R., Hope, J.M., Hinrichs, K.U., Hayes, J.M., Des Marais, D.J., 909 Cady, S.L., Summons, R.E., 2001. Signature lipids and stable carbon isotope analyses of The Sample Analysis at Mars Investigation and Instrument Suite. Space Sci. Rev. 1-78. McCollom, T.M., Hynek, B.M., Rogers, K.L., 2016. Potential for preservation of biosignatures 965 from endolithic microbial communities in a Mars analog fumarole environment, in: 966 Biosignature Preservation and Detection in Mars Analog Environments. p. 2006. Saez, C., Salthiel, S., Manga, M., Nguyen, C., Gonnermann, H., 2016. Physical and 977 hydraulic properties of modern sinter deposits: El Tatio, Atacama. J. Volcanol. Geotherm. Walter, M.R., Des Marais, D.J., 1993. Preservation of Biological Information in Thermal Spring 1076 Deposits: Developing a Strategy for the Search for Fossil Life on Mars. Icarus 101, 129-Zhang, C.L., Huang, Z., Li, Y.-L., Romanek, C.S., Mills, G.L., Gibson, R.A., Talbot, H.M., 1105 Wiegel, J., Noakes, J., Culp, R., White, D.C., 2007. Lipid Biomarkers, Carbon Isotopes, and 1106 Phylogenetic Characterization of Bacteria in California and Nevada Hot Springs.Figure 1. Environmental settings and facies of a generalized geothermal spring system. Modified from Campbell et al. (2015).Figure 2. Example TMAH thermochemolysis reaction with a carboxylic acid to form a methylated carboxylic acid. After the reaction, the molecule is more volatile and detectable to GC-MS. TMAH in methanol is available on the SAM and MOMA instruments.

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	899 900 901 902 903 904 905 955 956 984 1012 957 958 959 960 961 985 986 987 988 989 1013 1014 1015 1016 906 963 990 991	D i s t r i b u t i o n D i s t r i b u t i o n D i s t r i b u t i o n D i s t r i b u t i o n D i s t r i b u t i o n Lynch, K.L.K.L., 2017. Biosignature Preservation and Detection in Mars Analog Environments. Astrobiology 17, 363-400. https://doi.org/10.1089/ast.2016.1627 He, Y., Buch, A., Morisson, M., Szopa, C., Freissinet, C., Williams, A.J., Millan, M., Guzman, M., Gonzalez, R.N., Bonnet, J.Y., Coscia, D., Mahaffy, P., Johnson, S.S., 2019. Application of TMAH thermochemolysis to the detection of nucleobases: application to the MOMA and SAM space experiment. Talanta submitted. D i s t r i b u t D i s t r i b u t Martin, D.K., Maurer, J., McAdam, A.C., Mclennan, D., Nolan, T.J., Noriega, M., Pavlov, D Cay , The Bahamas. Geobiology 112-130. https://doi.org/10.1111/gbi.12196 D Rossi, A.P., Neukum, G., Pondrelli, M., van Gasselt, S., Zegers, T., Hauber, E., Chicarro, A., D D D D D D D A.A., Prats, B., Raaen, E., Sheinman, O., Sheppard, D., Smith, J., Stern, J.C., Tan, F., Trainer, M., Ming, D.W., Morris, R. V, Jones, J., Gundersen, C., Steele, A., Wray, J., Botta, O., Leshin, L.A., Owen, T., Battel, S., Jakosky, B.M., Manning, H., Squyres, S., Christopher, R.N., Francois, P.M., Sternberg, R., Buch, A., Sorensen, P., Coscia, D., Szopa, C., Teinturier, S., Baffes, C., Feldman, J., Flesch, G., Forouhar, S., Garcia, R., Keymeulen, D., Woodward, S., Block, B.P., Arnett, K., Miller, R., Edmonson, C., Gorevan, S., 2012. i s t r i b u t Oshima, M., Miyagawa, A., 1974. Comparative studies on the fatty acid composition of moderately and extremely thermophilic bacteria. Lipids 9, 476-480. https://doi.org/10.1007/BF02534274 Pancost, R.D., Pressley, S., Coleman, J.M., Benning, L.G., Mountain, B.W., 2005. Lipid i s t r i b u i i i i i i i Foing, B., 2008. Large-scale spring deposits on Mars? J. Geophys. Res. E Planets 113, 1-s t s t s t s t s t s t s t 17. https://doi.org/10.1029/2007JE003062 r i r i r i r i r i r i r i Ruff, S.W., Farmer, J.D., 2016. Silica deposits on Mars with features resembling hot spring biosignatures at El Tatio in Chile. Nat. Commun. 7, 13554. b u b u b u b u b u b u b u biomolecules in silica sinters: indicators of microbial biodiversity. Environ. Microbiol. 7, t t t t t t t t Herdianita, N.R., Browne, P.R.L., Rodgers, K.A., Campbell, K.A., 2000. Mineralogical and textural changes accompanying ageing of silica sinter. Miner. Depos. 35, 48-62. i o n i o n i o 66-77. https://doi.org/10.1111/j.1462-2920.2004.00686.x i o i o i o i o i o i o i o i o https://doi.org/10.1007/s11214-012-9879-z n Pancost, R.D., Pressley, S., Coleman, J.M., Talbot, H.M., Kelly, S.P., Farrimond, P., Schouten, n n n n n n n n
		27 29 30 31 32 33 34 35 36 38 39

888

Guignard, C., Lemee, L.,

Ambles, A., 2005

. Lipid constituents of peat humic acids and humin.

889

Distinction from directly extractable bitumen components using TMAH and TEAAc 890 thermochemolysis.

Org. Geochem. 36,[287][288][289][290][291][292][293][294][295][296][297]

Hays, L.E., Achenbach, L.A., Bailey, J., Barnes, R., Baross, J., Bertka, C., Boston, P.J., Boyd, 892 E.S., Cable, M., Chen, I., Ciesla, F., Des Marais, D.J., Domagal-Goldman, S.D., Cook, J.E., 893 Goldman, A., Hud, N., Laine, P., Lloyd, K., Lyons, T.W., Meadows, V.S., Mix, L.J., 894 Mojzsis, S.J., Muller, S., Pasek, M., Powell, M., Robinson, T., Rosenzweig, F., Schmidt, B., 895 Seelig, B., Springsteen, G., Vance, S., Welander, P., Williams, L., Wordsworth, R., 2015. 896 NASA Astrobiology Strategy 2015. 897 Hays, L.E.L.E., Graham, H.V.H.V., 962 976 Munoz-1002 Pumas, C., Pruetiworanan, S., Peerapornpisal, Y., 2018. Diatom diversity in some hot springs of 1003 northern Thailand. Botanica 24, 69-86. https://doi.org/https://doi.org/10.2478/botlit-2018-1004 0007 1005 Ray, P.H., White, D.C., Brock, T.D., 1971. Effect of temperature on the fatty acid composition 1006 of Thermus aquaticus. J. Bacteriol. 106, 25-30. 1007 Renaut, R.W., Jones, B., 2011. Hydrothermal environments, terrestrial, in: Reitner, J., Thiel, V. 1008 (Eds.), Encyclopedia of Geobiology. Springer, pp. 467-479. 1009 Renaut, R.W., Jones, B., Tiercelin, J.J., 1998. Rapid in situ silicification of microbes at Loburu 1010 hot springs, Lake Bogoria, Kenya rift valley. Sedimentology 45, 1083-1103. 1017 Ruff, S.W., Farmer, J.D., Calvin, W.M., Herkenhoff, K.E., Johnson, J.R., Morris, R. V, Rice, 1018 M.S., Arvidson, R.E., Bell III, J.F., Christensen, P.R., Squyres, S.W., 2011. Characteristics, 1019 distribution, origin, and significance of opaline silica observed by the Spirit rover in Gusev

Table 1 . Sample location, ID, depth, mineralogy, latitude, longitude, percent total carbon and percent total organic carbon.

 1

	Group	Sample ID	Sample	Sample Depth	Mineralogy	Latitude	Longitude	% TC	% TOC ±0.06
		IC160730.09.S	Vent deposit	0 cm	Opal-A, clinopyroxenes (e.g. augite, diopside), plagioclase, gypsum	N64°	W19°	0.24	0.24
		IC160730.09.I	Vent deposit	4 cm	Opal-A, plagioclase, diopside, magnesite	51.940'	33.513'	0.08	0.08
	Hveravellir Modern Active	IC160730.01.S IC160730.01.I	Mid-apron deposit Mid-apron deposit	0 cm 3 cm	Amorphous Amorphous	N64° 51.917'	W19° 33.437'	0.43 0.10	0.43 0.10
	Spring System	IC160731.08.S Distal apron deposit	0 cm	Amorphous			0.08	0.08
		IC160731.08.I1 Distal apron deposit	12 cm	Amorphous	N64° 51.911'	W19° 33.363'	0.13	0.13
		IC160731.08.I2 Distal apron deposit	18 cm	Amorphous			0.15	0.15
		IC160730.04.S	Pisolith layer	0 cm	Opal-A, plagioclase, magnesite	N64° 51.893'	W19° 33.564'	0.30	0.30
		IC160726.06.S	Vent deposit	0 cm	Opal-CT	N63°	W022°	0.02	0.02
		IC160726.06.I	Vent deposit	7 cm	Opal-CT	49.148'	41.159'	0.01	0.01
		IC160727.02.S	Vent deposit	0 cm	Opal-CT, halite, quartz	N63°	W022°	0.13	0.13
	Gunnuhver Modern Inactive Spring	IC160727.02.I	Vent deposit	5 cm	Opal-CT, halite, quartz	49.138'	41.168'	0.02	0.02
	System	IC160727.05.S	Mid-apron deposit	0 cm	Opal-CT	N63°	W022°	0.17	0.17
		IC160727.05.I	Mid-apron deposit	7 cm	Opal-CT	49.136'	41.149'	0.02	0.02
		IC160727.03.S Distal apron deposit	0 cm	Amorphous	N63°	W022°	0.15	0.15
		IC160727.03.I Distal apron deposit	7 cm	Amorphous	49.131'	41.205'	0.02	0.02
		IC160807.04.S	Vent deposit	0 cm	Amorphous, calcite	N64°	W023°	1.83	0.61
	Lýsuhóll	IC160807.04.I	Vent deposit	3 cm	Amorphous, calcite	50.651'	13.281'	0.85	0.18
	Relict Spring System	IC160809.03.I	Vent deposit	2 cm	Amorphous, minor calcite	N64° 50.650'	W023° 13.274'	0.61	0.43
		IC160808.12.S	Vent deposit	0 cm	Calcite	N64° 50.657'	W023° 13.177'	10.38	6.31

Table 1 .

 1 Sample location, ID, depth, mineralogy, latitude, longitude, percent total carbon and percent total organic carbon.

Table 3 con't. Comparison of flash and SAM-like ramp pyrolysis.

 3

	Group	Sample ID

Table 3 .

 3 Comparison of flash pyrolysis and SAM-like ramp pyrolysis of sinter samples. Green = FAME detection in both experiments.Yellow = FAME detection in flash pyrolysis experiment only. Orange = FAME detection in ramp pyrolysis experiment only. Blank cells = no FAME detection. Detection of the C 19 standard is included for completeness.

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Astrobiology F o r P e e r R e v i e w O n l y ; N o t f o r D i s t r i b u t i o n

Deport, C., Lemee, L., Ambles, A., 2006. Comparison between humic substances from soil and 826 peats using TMAH and TEAAc thermochemolysis. Org. Geochem. 37,[649][650][651][652][653][654][655][656][657][658][659][660][661][662][663][664] Eigenbrode, J.L., Summons, R.E., Steele, A., Freissinet, C., Millan, M., Navarro-González, R.,