Article Silica Precipitation in a Wet–Dry Cycling Hot Spring Chamber

Andrew Gangidine 1,*, Jeff R. Havig 2, Jeffrey S. Hannon 1 and Andrew D. Czaja 1

1 Department of , University of Cincinnati, Cincinnati, OH 45221, USA; [email protected] (J.S.H.); [email protected] (A.D.C.) 2 Department of and Environmental Sciences, University of Minnesota, Minneapolis, MN 55455, USA; [email protected] * Correspondence: [email protected] or [email protected]

 Received: 16 November 2019; Accepted: 9 January 2020; Published: 14 January 2020 

Abstract: Terrestrial hot springs have emerged as strong contenders for sites that could have facilitated the origin of life. Cycling between wet and dry conditions is a key feature of these systems, which can produce both structural and chemical complexity within protocellular material. Silica precipitation is a common phenomenon in terrestrial hot springs and is closely associated with life in modern systems. Not only does silica preserve evidence of hot spring life, it also can help it survive during life through UV protection, a factor which would be especially relevant on the . Determining which physical and chemical components of hot springs are the result of life vs. non-life in modern hot spring systems is a difficult task, however, since life is so prevalent in these environments. Using a model hot spring simulation chamber, we demonstrate a simple yet effective way to precipitate silica with or without the presence of life. This system may be valuable in further investigating the plausible role of silica precipitation in ancient terrestrial hot spring environments even before life arose, as well as its potential role in providing protection from the high surface UV conditions which may have been present on early Earth.

Keywords: hydrothermal; origin of life; early earth; hot spring; simulation chamber; silica; precipitation; model

1. Introduction In recent years, terrestrial (land-based) hot springs have gained substantial support as environments where life could have arisen on Earth (e.g., [1–3]). In particular, the ability for terrestrial hot springs to cycle between wet and dry conditions has been highlighted as a notable advantage over sub-aqueous hydrothermal systems and have been shown to possess the ability to promote or facilitate complex prebiotic chemical reactions including formation, polymerization, etc. [1,2]. As with most prebiotic science, it is often challenging to draw parallels between the ancient, lifeless Earth, and the modern life-abundant Earth. Terrestrial hot springs are no exception, as these environments contain ubiquitous life. Hot spring sources commonly house extremophilic microorganisms, outflow vents are colonized by both phototrophic and chemotrophic , and even siliceous hot spring deposits are home to endolithic microbial communities. These environments are inseparable from chemical traces of life, making it difficult to tease apart non-biologic components from biologically associated components. In many terrestrial hydrothermal systems, spring is supersaturated with respect to amorphous silica (SiO2)[4]. Silica is also thought to play an important role in synthesizing important prebiotic , specifically when subjected to dehydration and rehydration cycles [5–7] which can precipitate out of solution as the water drops with distance from the source.

Life 2020, 10, 3; doi:10.3390/life10010003 www.mdpi.com/journal/life Life 2020, 9, x FOR PEER REVIEW 2 of 9 associated with extant microbes (Figure 1) through silica-coatings [9–11]. While the exact functions/benefits of these coatings are not completely understood, they may provide some degree of UV protection as silica can shield from intense UV due to a high absorbance [12], a similar strategy employed by many microbial communities in hot spring systems which thrive as endolithic (within-rock) communities boring into the surface of siliceous sinter [13,14], or as hypolithic (under- rock) communities living under millimeters of loose siliceous sinter [15]. Life 2020, 10, 3 2 of 9 Here we present a relatively simple hot spring “simulation chamber” (Figure 2) which can be used to precipitate silica in geochemical conditions similar to those which could be found in modern Thishot spring silica precipitatesystems (e.g., can Table entomb 1). and This preserve system microbialprecipitates life silica [8], butthrough can also the be simple observed action to beof microscopeintimately associated slides being with attached extant to microbes a rotating (Figure cylinder1) through which is silica-coatings periodically submerged [ 9–11]. While into thea carboy exact offunctions synthesized/benefits hot ofspring these fluid coatings and arethen not exposed completely to air understood, within the theychamber. may provideThe fluid some is gravity degree fed of throughUV protection autoclavable as silica tubing can shield into fromthe polypropylen intense UV duee container to a high by radiation a carboy absorbance above the [12system.], a similar This systemstrategy can employed be used by to manyprecipitate microbial silica communities both with and in hotwithout spring the systems addition which of microbial thrive as organisms, endolithic (within-rock)and may prove communities useful for boringlaboratory-based into the surface experiments of siliceous seeking sinter [to13 investigate,14], or as hypolithic questions (under-rock) relating to thecommunities potential benefits living under of silica millimeters precipitation of loose in terrestrial siliceous sinterhot spring [15]. environments.

Figure 1. AA secondary electron imageimage (10(10 kV)kV) ofof a a microbial microbial filament filament collected collected from from an an outflow outflow stream stream of ofSteep Steep Cone, Cone, a circumneutral a circumneutral hot spring hot spring in Yellowstone in Yellowstone National National Park, displaying Park, displaying a coating ofa coating botryoidal of botryoidalsilica surrounding silica surrounding the filament the body. filament body.

2. MaterialsHere we and present Methods a relatively simple hot spring “simulation chamber” (Figure2) which can be used to precipitate silica in geochemical conditions similar to those which could be found in modernThe hotconstruction spring systems of this (e.g.,simulation Table1 chamber). This system was based precipitates on a prototype silica through design conceived the simple by action Prof. Davidof microscope Deamer slidesof UC beingSanta attachedCruz to facilitate to a rotating the polymerization cylinder which of is monomers periodically via submerged wet–dry cycling into a (Figurecarboy ofA1 synthesized in Appendix hot A). spring The fluidnewly and constructed then exposed apparatus to air within we used the chamber.for this study The fluidwas designed is gravity tofed simulate through a autoclavable modern hot tubingspring intomore the closely, polypropylene particularly container an outflow by a stream carboy where above life the is system. often Thisobserved system and can silica be used precipitation to precipitate is silicacommonplace. both with andThe withoutchamber the used addition the same of microbial rotating-cylinder organisms, technique,and may prove with useful some fornotable laboratory-based differences (Figure experiments 2). A seeking12 × 12 × to 0.25 investigate inch aluminum questions sheet relating served to the as potentialthe base of benefits the chamber, of silica precipitationin order to efficiently in terrestrial transfer hot spring heat from environments. a hot plate directly beneath the chamber. The walls of the chamber were constructed from 12 × 12 × 0.25 inch polycarbonate sheets affixed to the aluminum base,Table with 1.theSimulated top cover hot be springing affixed . with additional gasket material for sealing purposes. The cylinder was machined from polypropylene, with a 1-inch bore through the center in order to be mountedParameter on a 1-inch diameter rotating rod. Th Valuee rod was attached to a 1 RPM gear motor affixed through the outsidepH of the chamber. A polypropylene 7.67 container was placed directly below the rotating rod, so theTemperature cylinder could be partially submerged 50 ◦ Cwhen the container is filled with Conductivity 382 µS/cm fluid. Plastic clips were attached to the cylinder to mount 1 × 3 inch glass microscope slides so that they Salinity 0.19 ppt Total Dissolved Solids ~270 ppm Silica ~250–290 ppm Life 2020, 9, x FOR PEER REVIEW 3 of 9 could be rotated into the fluid within the container and out again as the motor spun the rod. A small vent with 47-mm membrane filter (0.45-µm pore size) was placed on top of the system to relieve while keeping the chamber clean from outside contaminants. Autoclavable tubing was used to deliver the simulated hot spring fluid from a polypropylene carboy mounted above the simulation chamber. The fluid would be added directly into the polypropylene container in the chamber via autoclavable tubing and could be removed from the container using a drain on the bottom side of the polypropylene container which was fed through the side of the chamber via autoclavable tubing. The completed chamber was assembled in a fume hood, and each component of the system was autoclaved

Lifeto ensure2020, 10 ,sterilization, 3 acid washed using a 10% solution of trace-metal grade (<1 ppb) nitric acid for3 of≥3 9 days, and triple-rinsed using 18.2 MΩ/cm deionized water.

Figure 2.2. TheThe hot hot spring spring simulation simulation chamber. chamber. (A) 3D (A rendering) 3D rendering of the simulation of the simulation chamber. ( Bchamber.) Photograph (B) Photographof the simulation of the chamber simulation mid-experiment. chamber mid-experiment. 2. Materials and Methods The simulated hot spring fluid used an extra-pure sodium silicate solution to serve as a silica source.The The construction solution ofwas this buffered simulation with chamber sodium was bicarbonate based on a prototypeand used designsodium conceived hydroxide by Prof.and hydrochloricDavid Deamer acid of UCin order Santa to Cruz achieve to facilitate a circumneutral the polymerization pH. The parameters of monomers set for via the wet–dry simulated cycling hot (Figurespring fluid A1 in used Appendix in theseA ).experiments The newly constructed(Table 1) were apparatus based off we geochemical used for this data study obtained was designed from Steep to Cone,simulate a circumneutral a modern hot hot spring spring more in closely,Yellowstone particularly Nation anal outflowPark. It should stream be where noted, life however, is often observed that hot springsand silica exhibit precipitation a wide isrange commonplace. of geochemical The chamberconditions used (e.g., the pH, same silica rotating-cylinder content, other technique,dissolved withspecies, some temperature, notable di ffconductivity;erences (Figure see2 [16]),). A 12so these12 conditions0.25 inch aluminumare by no means sheet exhaustive. served as the base × × of the chamber, in order to efficiently transfer heat from a hot plate directly beneath the chamber. The walls of the chamber wereTable constructed 1. Simulated from hot 12spring12 geochemistry.0.25 inch polycarbonate sheets affixed × × to the aluminum base, with theParameter top cover being affixed withValue additional gasket material for sealing purposes. The cylinder was machined from polypropylene, with a 1-inch bore through the center in pH 7.67 order to be mounted on a 1-inch diameter rotating rod. The rod was attached to a 1 RPM gear motor Temperature 50 °C affixed through the outside of the chamber. A polypropylene container was placed directly below Conductivity 382 µS/cm the rotating rod, so the cylinder could be partially submerged when the container is filled with fluid. Salinity 0.19 ppt Plastic clips were attached to the cylinder to mount 1 3 inch glass microscope slides so that they could Total Dissolved Solids × ~270 ppm be rotated into the fluid within the container and out again as the motor spun the rod. A small vent Silica ~250–290 ppm with 47-mm membrane filter (0.45-µm pore size) was placed on top of the system to relieve pressure whileTwo keeping experimental the chamber conditions clean from were outside used to contaminants. precipitate silica Autoclavable from the simulated tubing was hot used spring to deliver fluid. Onethe simulated involved hotonly spring silica fluidprecipitation from a polypropylene under sterile conditions, carboy mounted and the above other the involved simulation precipitation chamber. Thein the fluid presence would of be added added microbes directly (a into mix the of polypropylenecyanobacteria, Gleocapsa container, Oscillatoria in the chamber, Anabaena via autoclavable, Fischerella, andtubing Gleotrichia and could) which be removed were streaked from the onto container the 1 × using 3 inch a drainglass onmicroscope the bottom slides side before of the polypropylene being clipped container which was fed through the side of the chamber via autoclavable tubing. The completed chamber was assembled in a fume hood, and each component of the system was autoclaved to ensure sterilization, acid washed using a 10% solution of trace-metal grade (<1 ppb) nitric acid for 3 days, ≥ and triple-rinsed using 18.2 MΩ/cm deionized water. The simulated hot spring fluid used an extra-pure sodium silicate solution to serve as a silica source. The solution was buffered with sodium bicarbonate and used and hydrochloric acid in order to achieve a circumneutral pH. The parameters set for the simulated hot spring fluid used in these experiments (Table1) were based o ff geochemical data obtained from Steep Cone, a circumneutral hot spring in Yellowstone National Park. It should be noted, however, that hot springs exhibit a Life 2020, 10, 3 4 of 9 wide range of geochemical conditions (e.g., pH, silica content, other dissolved species, temperature, conductivity; see [16]), so these conditions are by no means exhaustive. Two experimental conditions were used to precipitate silica from the simulated hot spring fluid. One involved only silica precipitation under sterile conditions, and the other involved precipitation in the presence of added microbes (a mix of cyanobacteria, Gleocapsa, Oscillatoria, Anabaena, Fischerella, and Gleotrichia) which were streaked onto the 1 3 inch glass microscope slides before being clipped × to the rotating cylinder. In the second experiment, these microbes were chosen for being metabolically similar to phototrophic organisms commonly found in hot spring outflow vents. For both experiments, the simulated hot spring fluid was heated to 50 ◦C, a common temperature of phototrophic microbial colonized outflow streams in terrestrial hot springs.

Scanning electronLife 2020, 9, x FOR microscopy PEER REVIEW (SEM) was performed using a SCIOS Dual-Beam4Scanning of 9 Electron Microscope in the Advanced Materials Characterization Center at the University of Cincinnati. to the rotating cylinder. In the second experiment, these microbes were chosen for being metabolically Both secondarysimilar electron to phototrophic and Everhart-Thornley organisms commonly Detector found (i.e.,in hot “ET-detector”, spring outflow vents. a combination For both of secondary electron and back-scatteredexperiments, the electronsimulated detector)hot spring fluid imaging was heated was performedto 50 °C, a common under temperature high vacuum of using Pt/Au coating and voltagesphototrophic ranging microbial from community 5 to 10 colonized kV. outflow streams in terrestrial hot springs. Scanning electron microscopy (SEM) was performed using a SCIOS Dual-Beam Scanning Electron Microscope in the Advanced Materials Characterization Center at the University of 3. Results Cincinnati. Both secondary electron and Everhart-Thornley Detector (i.e., “ET-detector”, a combination of secondary electron and back-scattered electron detector) imaging was performed 3.1. Abiotic Experimentunder high vacuum using Pt/Au coating and voltages ranging from 5 to 10 kV. The first objective3. Results was to experimentally precipitate silica using the hot spring simulation chamber. Since silica commonly3.1. Abiotic interactsExperiment with life (e.g., via precipitation/deposition, coating organisms, etc.) in hydrothermal settings,The first while objective some was silica to experimentally precipitation precipitate may besilica biologically-influenced using the hot spring simulation (e.g., via metabolic re-precipitation),chamber. it is mostly Since silica thought commonly to occur interacts via abiologicwith life (e.g., processes via precipitation/deposition, such as cooling coating and dehydration [17]. organisms, etc.) in hydrothermal settings, while some silica precipitation may be biologically- Silica can precipitateinfluenced via (e.g., dehydration, via metabolic but re-precipitation), the relatively it is mostly fast rotation thought to (1 occur RPM) via abiologic combined processes with a temperature such as cooling and dehydration [17]. Silica can precipitate via dehydration, but the relatively fast of 50 ◦C and minimal venting meant the system would remain humid. Despite this, after seven days, rotation (1 RPM) combined with a temperature of 50 °C and minimal venting meant the system would micron-scale silicaremain precipitates humid. Despite were this, after observed, seven days, with micron-s thecale same silica precipitates characteristic were observed, botryoidal with the texture found in silica precipitatessame from characteristic modern botryoidal hot spring texture environments found in silica (Figure precipitates3) [ 9from–11 ,16modern]. hot spring environments (Figure 3) [9–11,16].

Figure 3. SEM imagery of botryoidal silica structures. (A) A secondary electron image (10 kV) of Figure 3. SEMbotryoidal imagery silica of precipitate botryoidal from a silicasinter sample structures. collected from (A Steep) A secondaryCone hot spring electronin Yellowstone image (10 kV) of botryoidal silicaNational precipitate Park. (B) fromET-detector a sinter imagesample (20 kV) of collectedsilica which precipitated from Steep on aCone microscope hot slide spring after in Yellowstone 1 week in the hot spring simulation chamber (abiotic), displaying the same botryoidal texture National Park. (B) ET-detector image (20 kV) of silica which precipitated on a microscope slide characteristic of silica precipitation. after 1 week in the hot spring simulation chamber (abiotic), displaying the same botryoidal texture characteristic3.2.of Added silica Microbial precipitation. Life Experiment The second objective was to simulate the coating of microbes with precipitated silica in hot 3.2. Added Microbialsprings. Life This Experimentwas accomplished by the addition of various cyanobacteria to the hot spring simulation chamber. A cyanobacteria mixture of Gleocapsa, Oscillatoria, Anabaena, Fischerella, and Gleotrichia The secondwas objective streaked wasonto microscope to simulate slides the attached coating to ofthe microbesrotating cylinder. with After precipitated seven days, silica the in hot springs. cyanobacteria were imaged via SEM and -dispersive X-ray (EDS) which showed This was accomplished by the addition of various cyanobacteria to the hot spring simulation chamber.

A cyanobacteria mixture of Gleocapsa, Oscillatoria, Anabaena, Fischerella, and Gleotrichia was streaked Life 2020, 10, 3 5 of 9

onto microscopeLife 2020, 9, slidesx FOR PEER attached REVIEW to the rotating cylinder. After seven days, the cyanobacteria5 of 9 were imaged via SEM and Energy-dispersive X-ray spectroscopy (EDS) which showed that the were that the bacteria were covered with/surrounded by botryoidal silica precipitate similar to that observed covered with/surrounded by botryoidal silica precipitate similar to that observed in microbial samples in microbial samples obtained from hot spring outflow streams in Yellowstone National Park (Figures obtained from4 and 5) hot [10,11,16]. spring outflow streams in Yellowstone National Park (Figures4 and5)[10,11,16].

Figure 4. SEMFigure imagery 4. SEM imagery of silica of silica precipitation precipitation and and microbial life. life. (A) (ET-detectorA) ET-detector image (5 image kV) of silica (5 kV) of silica precipitationprecipitation exhibited exhibited around around individual individual microbes microbes (white (white arrows) inin a Yellowstone a Yellowstone National National Park Park hot hot spring outflow stream. (B) ET-detector image (5 kV) of silica precipitation exhibited around spring outflow stream. (B) ET-detector image (5 kV) of silica precipitation exhibited around various various microbes (white arrows) which were added onto a microscope slide and run through the hot microbes (whitespring simulation arrows) chamber which werefor 1 addedweek. Silica onto precipitation a microscope can be slide observed and starting run through to cover the the hot spring simulationorganisms. chamber (C for) ET-detector 1 week. image Silica (5 precipitation kV) of a dense microbial can be observed mat collected starting from anto outflow cover stream the organisms. (C) ET-detectorin Yellowstone image (5National kV) of Park, a dense showing microbial silica precipitation. mat collected (D) ET-detector from an image outflow (10 kV) stream of a dense in Yellowstone microbial mat of various non-hot spring cyanobacteria added onto a microscope slide and run National Park, showing silica precipitation. (D) ET-detector image (10 kV) of a dense microbial mat of through the hot spring simulation chamber for 1 week, once again exhibiting silica precipitation various non-hotcoating the spring mat. cyanobacteria added onto a microscope slide and run through the hot spring simulationLife chamber 2020, 9, x FOR for PEER 1 week, REVIEW once again exhibiting silica precipitation coating the mat.6 of 9

Figure 5. SEMFigure imagery 5. SEM of imagery bacteria of bacteria run run through through the hot hot spring spring simulation simulation chamber, with chamber, subsequent with subsequent panels showing EDS mapping for carbon (co-localized with the bacteria), silicon, and (i.e., panels showingsilica). EDS mapping for carbon (co-localized with the bacteria), silicon, and oxygen (i.e., silica).

4. Discussion

4.1. Silica and Its Possible Role in the Origin of Life This wet–dry cycling simulation chamber can be used to produce abiotic silica precipitation without the need for total dehydration in relatively short periods of , and thus more accurately represents “natural” conditions that would be conducive to life. Thus, this system can even be used to study live organisms in a recreated hot spring setting, which could realistically survive throughout the course of an experiment. This allows for a unique, easy to assemble simulation chamber to produce and study silica precipitation with or without the addition life. When organisms are added to the system, it can successfully be used to at least partially replicate silica-coating observed in hot spring systems. The development of life on early Earth would likely have been a scenario fraught with obstacles. Natural conditions that may have aided in this process may play a vital role in the formation and subsequent colonization of life on Earth. UV conditions on early Earth are thought to be a key boundary for , and are largely dependent on various factors including atmospheric absorption, attenuation by water, and stellar variability [18,19]. The exact UV conditions throughout early Earth are still debated, and it is not yet known whether or not surface UV was hostile to life, or whether life may have exhibited some kind of defense against harmful UV, or instead dwelled in non-surface environments which would have been more shielded from UV [20,21]. If UV conditions on the early Earth were indeed harmful to life, silica precipitated from hydrothermal environments could have provided some protection from harmful UV while allowing the benefits of surface life [15]. By being able to precipitate silica through wet–dry cycling (hypothesized as being crucial for increasing complexity in prebiotic [1]), the simulation chamber we present here may be valuable in testing hypotheses regarding the ability of surface hot springs to shield from harmful UV radiation [15].

Life 2020, 10, 3 6 of 9

4. Discussion

4.1. Silica and Its Possible Role in the Origin of Life This wet–dry cycling simulation chamber can be used to produce abiotic silica precipitation without the need for total dehydration in relatively short periods of time, and thus more accurately represents “natural” conditions that would be conducive to life. Thus, this system can even be used to study live organisms in a recreated hot spring setting, which could realistically survive throughout the course of an experiment. This allows for a unique, easy to assemble simulation chamber to produce and study silica precipitation with or without the addition life. When organisms are added to the system, it can successfully be used to at least partially replicate silica-coating observed in hot spring systems. The development of life on early Earth would likely have been a scenario fraught with obstacles. Natural conditions that may have aided in this process may play a vital role in the formation and subsequent colonization of life on Earth. UV conditions on early Earth are thought to be a key boundary for abiogenesis, and are largely dependent on various factors including atmospheric absorption, attenuation by water, and stellar variability [18,19]. The exact UV conditions throughout early Earth are still debated, and it is not yet known whether or not surface UV was hostile to life, or whether life may have exhibited some kind of defense against harmful UV, or instead dwelled in non-surface environments which would have been more shielded from UV [20,21]. If UV conditions on the early Earth were indeed harmful to life, silica precipitated from hydrothermal environments could have provided some protection from harmful UV while allowing the benefits of surface life [15]. By being able to precipitate silica through wet–dry cycling (hypothesized as being crucial for increasing complexity in prebiotic chemistry [1]), the simulation chamber we present here may be valuable in testing hypotheses regarding the ability of surface hot springs to shield from harmful UV radiation [15].

4.2. Wet–Dry Cycling Understanding the transition of the Earth from a lifeless to a life-filled world has been a question at the forefront of science, notably address in Charles Darwin’s letter to his friend J. D. Hooker in 1871, where he supposed a “warm little pond” where life may have originated [22]. In recent years, Darwin’s “warm little pond” hypothesis for the origin of life has gained support, with researchers attempting to recreate such conditions in the lab. To this day, laboratory-based simulation chambers attempt to simulate the prebiotic Earth. Early experimentation showed the value of wet–dry cycling, whereupon liposomes subjected to wet–dry cycling resulted in the formation of multilamellar structures trapping solute during drying phases, and the lamellae-forming vesicles containing solute during rehydration [23]. Recent advances have built upon this concept, showing that molecular systems subjected to hydration and dehydration cycles in prebiotic analogue systems undergo chemical (e.g., polymerization), encapsulation, and combinatorial selection [1]. These results have been replicated by other laboratories equipped with instrumentation which also supports wet–dry cycling, such as a new “ simulator” with a similar “” added into it, to simulate seasons in a volcanic surface environment (i.e., hot during the day yet cold at night). The simulator eventually showed the formation of -like structures which incorporated genetic material, similar to what was observed in prior wet–dry experiments [24]. Other laboratory-based wet–dry cycling experiments have likewise been used to suggest that wet–dry cycling could be used to form polypeptides (key components of modern life) on the prebiotic Earth [25]. Through such laboratory-based wet–dry cycling experimental work, terrestrial hot springs have emerged as convincing environments which may have harbored the origin of life on Earth and will undoubtedly continue to be investigated. We propose our new wet–dry cycling chamber as an additional tool to test the validity of hypotheses involving hydrothermal systems and wet–dry cycling, specifically those which propose an important role for silica precipitation. Life 2020, 10, 3 7 of 9

4.3. Variation and Future Studies Hot spring systems are complex and highly variable [16]. Even the same spring can vary wildly in terms of geochemistry from one year to the next [26,27]. Several variations of this system could be attempted in the future to test more specific hypotheses such as running the system under anoxic atmospheric conditions which may more closely resemble conditions of the ancient Earth, testing a wider range of geochemical conditions (e.g., more acidic, more alkaline, different element concentrations), testing various , and adding other microorganisms. By testing such variables, we may better understand terrestrial hot spring systems and the vast roles they have been hypothesized to play in potentially facilitating the origin, evolution, and dispersal of life on the early Earth (e.g., [1,12]).

Author Contributions: Conceptualization, A.G., methodology, A.G., J.R.H. and A.D.C.; validation, A.G., J.R.H. and A.D.C.; formal analysis, A.G. and J.S.H.; writing—original draft preparation, A.G.; writing—review and editing, J.R.H., J.S.H. and A.D.C.; visualization, A.G.; supervision, A.D.C.; project administration, J.R.H. and A.D.C.; funding acquisition, A.G. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the University of Cincinnati chapter of Sigma Xi, as well as the Ohio Grant Consortium. Acknowledgments: We thank David Deamer and Bruce Damer for their help in the design and development of this simulation chamber. We also wish to thank Andrew Volz and Michael Menard of the University of Cincinnati for their help in assembling the simulation chamber. Yellowstone National Park samples were collected and analyzed in accordance with Yellowstone National Park Research Permit #YELL-2017-SCI-7020 held by J.R.H. Conflicts of Interest: The authors declare no conflict of interest.

Appendix A Life 2020, 9, x FOR PEER REVIEW 8 of 9

Appendix A

Figure A1. An earlyFigure wet–dryA1. An early cyclingwet–dry cycling apparatus apparatus protot prototypeype developed developed by Prof. David by Deamer Prof. to David Deamer to promote monomer polymerization. This apparatus consists of a hollow stainless-steel cylinder, which promote monomerrotates polymerization. once per hour via the This minute apparatus hand of a clock consists motor. The lower of a portion hollow of the stainless-steel cylinder rotates cylinder, which rotates once per hourinto a reaction via the mixture minute placed hand into the ofdish a below. clock A heating motor. element The is lower used to heat portion the cylinder of theto cylinder rotates 80°C. CO2 or N2 enters the apparatus at a low rate (<5 ccs/s) to carry off the water produced by the into a reaction mixturecondensation placed reactions into that the polymerize dish mononucleo below. Atides. heating A typical element reaction mixture is used holds 10 to mM heat the cylinder to 80 C. CO or N entersmononucleotides the apparatus in their acid form, at together a low with rate either (< 105 ccsmM /phospholipids) to carry or 100 off mMthe water produced by the ◦ 2 2 chloride, both of which promote polymerization. As the cylinder rotates out of the reaction mixture, condensation reactionsa thin film that of the polymerizemixture is picked up mononucleotides. on the burnished surface of A the typical cylinder and reaction would be quickly mixture holds 10 mM mononucleotidesdried in their by the acidheat so form, that the togethermonomers could with poly eithermerize. The 10 dried mM film phospholipid then re-dissolves when or 100the mM ammonium cylinder rotates back into the mixture. To prevent the entire 15 mL from evaporating, a perfusion chloride, both of whichpump delivers promote water at polymerization.the same rate it evaporated. As the cylinder rotates out of the reaction mixture,

a thin film of theReferences mixture is picked up on the burnished surface of the cylinder and would be quickly

dried by the heat1. Damer, so that B.; theDeamer, monomers D. Coupled phases could and polymerize.combinatorial selection The in dried fluctuating film hydrothermal then re-dissolves pools: A when the cylinder rotates backscenario into to guide the mixture.experimental Toapproaches prevent to the the origin entire of cellular 15 life. mL Life from 2015, 5 evaporating,, 872–887. a perfusion pump 2. Milshteyn, D.; Damer, B.; Havig, J.; Deamer, D. Amphiphilic compounds assemble into membranous delivers water at thevesicles same in hydrothermal rate it evaporated. hot spring water but not in seawater. Life 2018, 8, 11. 3. Deamer, D.; Damer, B.; Kompanichenko, V. Hydrothermal chemistry and the origin of cellular life. 2019, 19, 1523–1537. 4. White, D.E.; Brannock, W.W.; Murata, K.J. Silica in hot-spring . Geochim. Cosmochim. Acta 1956, 10, 27–59. 5. Schwartz, A.W.; Chittenden, G.J.F. Synthesis of uracil and thymine under simulated prebiotic conditions. Biosystems 1977, 9, 87–92. 6. Hargreaves, W.R.; Mulvihill, S.J.; Deamer, D.W. Synthesis of phospholipids and membranes in prebiotic conditions. 1977, 266, 78. 7. Kitadai, N.; Oonishi, H.; Umemoto, K.; Usui, T.; Fukushi, K.; Nakashima, S. polymerization on oxide minerals. Orig. Life Evol. Biosph. 2017, 47, 123–143. 8. Alleon, J.; Bernard, S.; Le Guillou, C.; Daval, D.; Skouri-Panet, F.; Pont, S.; Delbes, L.; Robert, F. Early entombment within silica minimizes the molecular degradation of microorganisms during advanced diagenesis. Chem. Geol. 2016, 437, 98–108.

Life 2020, 10, 3 8 of 9

References

1. Damer, B.; Deamer, D. Coupled phases and combinatorial selection in fluctuating hydrothermal pools: A scenario to guide experimental approaches to the origin of cellular life. Life 2015, 5, 872–887. [CrossRef] 2. Milshteyn, D.; Damer, B.; Havig, J.; Deamer, D. Amphiphilic compounds assemble into membranous vesicles in hydrothermal hot spring water but not in seawater. Life 2018, 8, 11. [CrossRef] 3. Deamer, D.; Damer, B.; Kompanichenko, V.Hydrothermal chemistry and the origin of cellular life. Astrobiology 2019, 19, 1523–1537. [CrossRef][PubMed] 4. White, D.E.; Brannock, W.W.; Murata, K.J. Silica in hot-spring waters. Geochim. Cosmochim. Acta 1956, 10, 27–59. [CrossRef] 5. Schwartz, A.W.; Chittenden, G.J.F. Synthesis of uracil and thymine under simulated prebiotic conditions. Biosystems 1977, 9, 87–92. [CrossRef] 6. Hargreaves, W.R.; Mulvihill, S.J.; Deamer, D.W. Synthesis of phospholipids and membranes in prebiotic conditions. Nature 1977, 266, 78. [CrossRef][PubMed] 7. Kitadai, N.; Oonishi, H.; Umemoto, K.; Usui, T.; Fukushi, K.; Nakashima, S. Glycine polymerization on oxide minerals. Orig. Life Evol. Biosph. 2017, 47, 123–143. [CrossRef][PubMed] 8. Alleon, J.; Bernard, S.; Le Guillou, C.; Daval, D.; Skouri-Panet, F.; Pont, S.; Delbes, L.; Robert, F. Early entombment within silica minimizes the molecular degradation of microorganisms during advanced diagenesis. Chem. Geol. 2016, 437, 98–108. [CrossRef] 9. Handley, K.M.; Turner, S.J.; Campbell, K.A.; Mountain, B.W. Silicifying biofilm exopolymers on a hot-spring microstromatolite: Templating nanometer-thick laminae. Astrobiology 2008, 8, 747–770. [CrossRef] 10. Smythe, W.F.; McAllister, S.M.; Hager, K.W.; Hager, K.R.; Tebo, B.M.; Moyer, C.L. Silica Biomineralization of Calothrix-Dominated Biofacies from Queen’s Laundry Hot-Spring, Yellowstone National Park, USA. Front. Environ. Sci. 2016, 4, 40. [CrossRef] 11. Gangidine, A.; Jeff, R.H.; David, A.F.; Jones, C.; Hamilton, T.L.; Czaja, A.D. Trace element concentrations in hydrothermal silica deposits as a potential biosignature. Astrobiology 2020, 20, 1994. [CrossRef][PubMed] 12. Phoenix, V.R.; Bennett, P.C.; Engel, A.S.; Tyler, S.W.; Ferris, F.G. Chilean high-altitude hot-spring sinters: A model system for UV screening mechanisms by early Precambrian cyanobacteria. Geobiology 2006, 4, 15–28. [CrossRef] 13. Gaylarde, P.M.; Jungblut, A.-D.; Gaylarde, C.C.; Neilan, B.A. Endolithic phototrophs from an active geothermal region in New Zealand. Geomicrobiol. J. 2006, 23, 579–587. [CrossRef] 14. Walker, J.J.; Spear, J.R.; Pace, N.R. Geobiology of a microbial endolithic community in the Yellowstone geothermal environment. Nature 2005, 434, 1011. [CrossRef][PubMed] 15. Havig, J.R.; Hamilton, T.L. Hypolithic in hydrothermal areas and implications for cryptic oxygen oases on Archean continental surfaces. Front. Earth Sci. 2019, 7, 15. [CrossRef] 16. Havig, J.R.; Kuether, J.; Gangidine, A.; Schroeder, S.; Hamilton, T.L. Hot Spring Microbial Community Elemental Composition: Hot Spring and Soil Inputs, and the Transition from Biocumulus to Siliceous Sinter. Astrobiology 2019. in revision. 17. Guidry, S.A.; Chafetz, H.S. Factors governing subaqueous siliceous sinter precipitation in hot springs: Examples from Yellowstone National Park, USA. Sedimentology 2002, 49, 1253–1267. [CrossRef] 18. Ranjan, S.; Sasselov,D.D. Influence of the UV environment on the synthesis of prebiotic molecules. Astrobiology 2016, 16, 68–88. [CrossRef] 19. Ranjan, S.; Sasselov, D.D. Constraints on the early terrestrial surface UV environment relevant to prebiotic chemistry. Astrobiology 2017, 17, 169–204. [CrossRef] 20. Westall, F.; De Ronde, C.E.; Southam, G.; Grassineau, N.; Colas, M.; Cockell, C.; Lammer, H. Implications of a 3.472–3.333 Gyr-old subaerial microbial mat from the Barberton greenstone belt, South Africa for the UV environmental conditions on the early Earth. Philos. Trans. R. Soc. B Biol. Sci. 2006, 361, 1857–1876. [CrossRef] 21. Westall, F.; Hickman-Lewis, K.; Hinman, N.; Gautret, P.; Campbell, K.A.; Bréhéret, J.-G.; Foucher, F.; Hubert, A.; Sorieul, S.; Dass, A.V.; et al. A hydrothermal-sedimentary context for the origin of life. Astrobiology 2018, 18, 259–293. [CrossRef][PubMed] 22. Darwin, C. Darwin Correspondence Project, “Letter No. 7471”. 1871. Available online: http://www. darwinproject.ac.uk/DCP-LETT-7471 (accessed on 10 December 2019). 23. Deamer, D.W.; Barchfeld, G.L. Encapsulation of by vesicles under simulated prebiotic conditions. J. Mol. Evol. 1982, 18, 203–206. [CrossRef][PubMed] Life 2020, 10, 3 9 of 9

24. Casey, L. McMaster University Researchers Testing Origins of Life Theory in New Planet Simulator; Canadian Press: Toronto, ON, Canada, 2018. 25. Forsythe, J.G.; Yu, S.-S.; Mamajanov, I.; Grover, M.A.; Krishnamurthy, R.; Fernández, F.M.; Hud, N.V. Ester-mediated amide bond formation driven by wet–dry cycles: A possible path to polypeptides on the prebiotic Earth. Angew. Chem. Int. Ed. 2015, 54, 9871–9875. [CrossRef][PubMed] 26. Ball, J.W.; McCleskey, R.B.; Nordstrom, D.K.; Holloway, J.M. Water-Chemistry Data for Selected Springs, Geysers, and Streams in Yellowstone National Park, Wyoming, 2003–2005; Geological Survey: Reston, VA, USA, 2008. 27. Ball, J.W.; McMleskey, R.B.; Nordstrom, D.K. Water-Chemistry Data for Selected Springs, Geysers, and Streams in Yellowstone National Park, Wyoming, 2006–2008; US Geological Survey: Reston, VA, USA, 2010.

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).