Chemical Gardens as Flow-through Reactors Simulating Natural Hydrothermal Systems

Podsumowanie

We describe chemical garden formation via injection experiments that allow for laboratory simulations of natural chemical garden systems that form at submarine hydrothermal vents.

Streszczenie

Here we report experimental simulations of hydrothermal chimney growth using injection chemical garden methods. The versatility of this type of experiment allows for testing of various proposed ocean / hydrothermal fluid chemistries that could have driven reactions toward the origin of life in environments on the early Earth, early Mars, or even other worlds such as the icy moons of the outer planets. We show experiments that include growth of chemical garden structures under anoxic conditions simulating the early Earth, inclusion of trace components of phosphates / organics in the injection solution to incorporate them into the structure, a switch of the injection solution to introduce a secondary precipitating anion, and the measurement of membrane potentials generated by chemical gardens. Using this method, self-assembling chemical garden structures were formed that mimic the natural chimneys precipitated at submarine hydrothermal springs, and these precipitates can be used successfully as flow-through reactors by feeding through multiple successive “hydrothermal” injections.

Wprowadzenie

“Chemical gardens” are self-assembling inorganic precipitates developed where two fluids of contrasting chemistries interact^1,2. These self-assembling inorganic structures have been the subject of scientific interest for over a century partly due to their biomimetic appearance, and many experimental and theoretical studies have been pursued to understand the various complex aspects and possible functions of chemical garden systems³. Natural examples of chemical gardens include mineral “chimney” precipitates that grow around hydrothermal springs and seeps, and it has been argued that these could provide plausible environments for life to emerge⁴. To grow a chemical garden simulating a natural hydrothermal vent chimney, a reservoir solution should represent a simulated ocean composition and an injection solution should represent the hydrothermal fluid that feeds into the ocean. The versatility of this type of experiment to different reaction systems allows for simulation of almost any proposed ocean / hydrothermal fluid chemistry, including environments on the early Earth or on other worlds. On the early Earth, the oceans would have been anoxic, acidic (pH 5-6), and would have contained dissolved atmospheric CO₂ and Fe²⁺, as well as Fe^III, Ni²⁺, Mn²⁺, NO^3-, and NO^2-. Chemical reactions between this seawater and the ultramafic ocean crust would have produced an alkaline hydrothermal fluid containing hydrogen and methane, and in some cases sulfide (HS^-)^4-8. The chimneys formed in early Earth alkaline vent environments could thus have contained ferrous/ferric oxyhydroxides and iron/nickel sulfides, and it has been proposed that these minerals might have served particular catalytic and proto-enzymatic functions toward harnessing geochemical redox / pH gradients to drive the emergence of metabolism⁵. Likewise, on other worlds such as that may host (or may have hosted) water/rock interfaces — such as early Mars, Jupiter’s moon Europa, or Saturn’s moon Enceladus — it is possible that water/rock chemistry could generate alkaline vent environments capable of driving prebiotic chemistry or even providing habitable niches for extant life^5,9-11.

The classic chemical garden experiment involves a seed crystal of a metal salt, e.g. ferrous chloride tetrahydrate FeCl₂•4H₂O, submerged in a solution containing reactive anions, e.g. sodium silicate or “water glass”. The metal salt dissolves, creating an acidic solution containing Fe²⁺ that interfaces with the more alkaline solution (containing silicate anions and OH^-) and an inorganic membrane precipitate is formed. The membrane swells under osmotic pressure, bursts, then re-precipitates at the new fluid interface. This process repeats until the crystals are dissolved, resulting in a vertically oriented, self-organized precipitate structure with complex morphology at both macro and micro scales. This precipitation process results in the continued separation of chemically contrasting solutions across the inorganic chemical garden membrane, and the difference of charged species across the membrane yields a membrane potential^12-14. Chemical garden structures are complex, exhibiting compositional gradients from interior to exterior^13,15-19, and the walls of the structure maintain separation between contrasting solutions for long periods while remaining somewhat permeable to ions. In addition to being an ideal experiment for educational purposes (as they are simple to make for classroom demonstrations, and can educate students about chemical reactions and self-organization), chemical gardens have scientific significance as representations of self-assembly in dynamic, far-from-equilibrium systems, involving methods that can lead to the production of interesting and useful materials^20,21.

Chemical gardens in the laboratory can also be grown via injection methods, in which the solution containing one precipitating ion is slowly injected into the second solution containing the co-precipitating ion (or ions). This results in the formation of chemical garden structures similar to those of crystal growth experiments, except that the properties of the system and the precipitate can be better controlled. The injection method has several significant advantages. It allows one to form a chemical garden using any combination of precipitating or incorporated species; i.e., multiple precipitating ions can be incorporated into one solution, and/or other non-precipitating components can be included in either solution to adsorb / react with the precipitate. The membrane potential generated in a chemical garden system can be measured in an injection experiment if an electrode is incorporated into the interior of the structure, thus enabling electrochemical study of the system. Injection experiments offer the ability to feed the injection solution into the interior of the chemical garden for controlled time frames by varying the injection rate or total injected volume; it is therefore possible to feed through different solutions sequentially and use the precipitated structure as a trap or reactor. Combined, these techniques allow for laboratory simulations of the complex processes that could have occurred in a natural chemical garden system at a submarine hydrothermal vent, including a chimney formed from many simultaneous precipitation reactions between ocean and vent fluid (e.g., producing metal sulfides, hydroxides, and/or carbonates and silicates)^5,22. These techniques can also be applied to any chemical garden reaction system to allow for formation of new types of materials, e.g., layered tubes or tubes with adsorbed reactive species^20,23.

We detail here an example experiment that includes the simultaneous growth of two chemical gardens, Fe²⁺-containing structures in an anoxic environment. In this experiment we incorporated trace amounts of polyphosphates and/or amino acids into the initial injection solution to observe their effect on the structure. After initial formation of the chemical garden we then switched the injection solution to introduce sulfide as a secondary precipitating anion. Measurements of membrane potentials were made automatically throughout the experiment. This protocol describes how to run two experiments at once using a dual syringe pump; the data shown required multiple runs of this procedure. The relatively high flow rates, low pH of the reservoir and reactant concentrations employed in our experiments are designed to form large chimney precipitates on time scales suitable for one-day laboratory experiments. However, fluid flow rates at natural hydrothermal springs can be much more diffuse and the concentrations of precipitating reactants (e.g., Fe and S in an early Earth system) could be an order of magnitude lower⁴; thus, structured precipitates would form over longer timescales and the vent could be active for tens of thousands of years^24,25.

Access restricted. Please log in or start a trial to view this content.

Protokół

1. Safety Considerations

1. Use personal protective equipment (lab coat, goggles, nitrile gloves, proper shoes) to prevent against chemical spills or injury. Use syringes and needles, and take care to not puncture gloves. Take care during experiment setup to check the apparatus for leaks by performing the injection first with double distilled H₂O (ddH₂O), and to check the stability of the reaction vials on the stand, before adding chemicals.
2. Undertake this experiment with any chemical garden recipe, but one of the reactants we use to simulate deep-sea vents is a hazardous chemical, sodium sulfide; therefore do the entire experiment inside a fume hood to prevent exposure.
   1. Only open the bottle of sodium sulfide in the fume hood and place a balance inside the fume hood for weighing sulfide. Always keep sulfide-containing solutions inside the fume hood as they release toxic H₂S gas, and also keep sulfide liquid, sharps, and solid waste containers in the fume hood. Another reactant of interest is Fe(II)Cl₂•4H₂O, which oxidizes upon exposure to air, so take care to keep solutions anoxic and to grow chemical gardens under an anoxic headspace (e.g., N₂ or Ar), always within a fume hood or glove box.

2. Setup for Injection Experiments

1. Create glass “injection vials” by cutting off the bottom 1 cm of a 100 ml clear glass crimp top serum bottle (20 mm crimp seal closure type) with a glass cutter so that, when inverted, the vessel is open to the air. As these are reusable, clean the vials in a 1 M HCl acid bath O/N, and then rinse well with ddH₂O before a new experiment.
2. Prepare the injection vials (Figure 1).
   1. Collect a 20 mm septum, 20 mm aluminum crimp seal, and a 0.5-10 µl plastic pipette tip. Using a 16 G syringe needle, carefully puncture a hole through the center of the septum, then remove and discard the needle in the appropriate sharps waste container.
   2. Insert the pipette tip into the needle hole, into the side of the rubber septum that will face inside the crimp top of the vial. Push the pipette tip through the septum so that it pokes out the other side.
   3. Crimp-seal the septum with pipette tip onto the injection vessel to make a watertight seal. When sealed, push the pipette tip further through the septum so that it protrudes outside.
   4. Affix 1/16” inner diameter clear flexible chemical-resistant tubing to the pipette tip (tubing length should reach from the injection vial to the syringe pump); slide it up for a watertight seal.
      Note: This will be the injection tube, fed from the other end by a syringe with 16 G needle.
   5. Check for leaks: Insert a 10 ml syringe filled with ddH₂O with a 16 G needle into the other end of the tubing (smoothly slide the tubing straight onto the needle and be careful not to puncture the wall of the tubing). Slowly inject so that the ddH₂O moves up the tubing and into the bottom of the reaction vessel. Ensure that the syringe/tube, tube/tip, and crimp seals are watertight.
3. Clamp the injection vials on a stand in a fume hood, so that the injection will feed in from the bottom of the vial.
   Note: Multiple vials can be set up at once and fed simultaneously by separate syringes.
4. Set up electrodes for measuring membrane potential across the wall of the chemical gardens. Always use the same convention for which lead is “inside” and which is “outside” of the chemical gardens.
   1. Cut lengths of insulated wire (e.g., copper) that reach from inside the reaction vessels to the lead of the multimeter or data logger. Leave a little bit of slack in the wires for positioning.
   2. Strip ~3 mm of the wire bare at the ends that will be located inside the reaction vial. At the other ends that will be connected to the multimeter leads, strip ~1 cm of wire.
   3. Fix the wires in place to measure membrane potential across the chemical garden. For the wire that will go inside the chemical garden: insert it into the opening of the pipette tip from which fluid will feed into the vessel.
   4. Push the wire in lightly to ensure contact with the injection solution, but not so far that it will clog the injection flow. For the outside wire: place it so that it will be in contact with the solution reservoir but not with the chemical garden precipitate.
   5. Tape or otherwise secure the wires so that they cannot move inside the injection vial during the experiment (Figure 2).
   6. Attach the other ends of the wires to the multimeter, and secure the wires so that those ends also do not move throughout the experiment.
5. Set up N₂ gas lines that will each feed into one of the injection vials.
   1. Split the gas feed from a N₂ source into several tubes, so that there is one N₂ feed for each injection vial.
   2. Place each N₂ tube so that it feeds into the headspace of one of the injection vials.

3. Preparation of Solutions for Chemical Garden Growth

1. Prepare the reservoir solution, 100 ml for each experiment. Note: In this example, use 75 mM Fe²⁺ and 25 mM Fe³⁺ as the precipitating cations (Table 1).
   1. Create anoxic solutions by first bubbling the ddH₂O with N₂ gas for ~15 min per 100 ml.
   2. Weigh out and add the FeCl₂•4H₂O and FeCl₃•6H₂O, stirring gently to dissolve (not vigorously so as to not introduce oxygen).
   3. After reagents are dissolved, immediately resume light bubbling of the Fe²⁺/Fe³⁺ solution with N₂ gas while injections are prepared.
2. Choose any two of the primary injection solutions shown in Table 1, and prepare 10 ml of each. Fill a 10 ml syringe to the 7 ml mark with each of the solutions (one syringe for each solution). Replace the needle caps and set aside.
3. Prepare 20 ml of the secondary injection solution (sodium sulfide — CAUTION) shown in Table 1. Fill two 10 ml syringes to the 7 ml mark with this solution, replace the needle caps and set aside. Always keep sulfide-containing solutions and syringes in the fume hood.
4. Refill the ddH₂O syringes from Step 2.2.5; these will be used to flush the injection tube.

4. Starting the Primary Injection

1. Use desired data logger for membrane potential measurements; measure each experiment’s potential on a separate channel, and set the scan rate to give the desired amount of data points (e.g., for a 2-hr injection, recording potential every 30 sec would be sufficient).
2. Secure the primary injection syringes on the programmable syringe pump in the fume hood.
3. Use a waste beaker to catch drips and set the syringe pump to inject at a fast rate until the syringes both begin to drip into the beaker. Then stop the injection (in order to ensure that the two syringes begin injecting at exactly the same level).
4. Re-program the syringe pump to inject at 2 ml per hour (calibrate for the type of syringe being used), but do not hit start.
5. Insert the ddH₂O syringes into the two plastic injection tubes, and inject so that the water fills the clear tubing up to the aperture where it enters the main reservoir. Place the syringes on the stand, above the injection vials.
6. Pour 100 ml of the Fe²⁺/Fe³⁺ reservoir solution into each vial.
7. Adjust the flow of the N₂ gas lines as desired to keep the experiment anoxic for the duration of the injections.
8. Carefully cover the reservoir vials with an airtight seal (e.g., using Parafilm; not obstructing the view through the glass) and insert an N₂ feed into each vial (Figure 3).
9. Bring the ddH₂O syringes (still inserted in the tubing) down next to the primary injection syringes. Carefully slide the plastic injection tubing off the ddH₂O syringe needle, and immediately transfer it directly onto one of the primary injection syringe needles. (Take care to not puncture the wall of the tubing.)
10. Start the injection, and start recording of membrane potential.

5. Starting the secondary injection:

1. Hit stop on the syringe pump after 3 hr (after 6 ml have been injected), once chemical garden structures have formed (Figure 4), continually generating a membrane potential (Figure 5).
2. Carefully remove the primary injection syringes from the syringe pump (but leave them connected to the tubing so the structures are not disturbed); set them on the stand above the level of the fluid in the vials so that the fluid cannot flow back into the syringe.
3. Secure the secondary injection sulfide syringes to the syringe pump, and repeat Steps 4.3 and 4.4.
4. Remove the secondary syringes one at a time from the syringe pump, and, while holding the syringes above the level of the fluid in the vials, repeat Step 4.9, transferring the tubing from the primary syringes to the secondary syringes (Figure 6). Be vigilant that the fluid pressure from the reservoir into the syringe does not cause fluid to flow back into the syringe as this could collapse the chemical garden.
5. When the transfer is complete, carefully secure the secondary syringes to the syringe pump.
6. Re-program the syringe pump to inject at 2 ml per hour, and hit start to continue the injection with the new injection solution.
7. Safely dispose of the primary injection syringes.

6. Ending the Experiment

1. First stop the syringe pump, then stop recording of the membrane potential and save the data.
2. Turn off the N₂ flow and remove the lines and the Parafilm from the injection vessels.
3. If desired, sample the reservoir solution or precipitate for further analysis. To carefully remove the reservoir solution and not disturb the precipitate, use a 25 ml pipette to carefully pipette off the reservoir solution in several aliquots, and discard the solution in a waste beaker.
4. Unclamp the injection vessels one at a time and pour the solution into a waste transfer beaker in the fume hood. Use ddH₂O to rinse out pieces of precipitate.
5. Remove the syringes from the syringe pump, and extract them from the tubing, letting extra injection fluid run off into the waste transfer beaker. Empty the syringes into the waste beaker, and dispose of the syringes in a sulfide sharps container kept in the fume hood.
6. Remove the tubing from the experiment vial and dispose of it in a solid waste bag. Uncrimp the seal and dispose of the septum, seal, and pipette tip.
7. Rinse out the glass experiment vial and soak it in a 1 M HCl acid bath O/N. (CAUTION — glassware that has been in contact with sodium sulfide will release toxic H₂S gas when placed in acid. Keep acid baths inside the fume hood.)

Access restricted. Please log in or start a trial to view this content.

Wyniki

Once the injection solution started to feed into the reservoir solution, a chemical garden precipitate began to form at the fluid interface and this structure continued to grow over the course of the injection (Figures 4-7). In the experiments reported here, the first injection was sodium hydroxide (which can be modified to include L-alanine and/or pyrophosphate), and the reservoir solution was a 1:3 mixture of Fe³⁺/Fe²⁺, yielding a mixed-redox-state iron oxyhydroxide precipitate. T...

Access restricted. Please log in or start a trial to view this content.

Dyskusje

The formation of a chemical garden structure via injection method can be accomplished by interfacing any two solutions containing reactive ions that produce a precipitate. There are many possible reaction systems that will produce precipitate structures and finding the right recipe of reactive ions and concentrations to grow a desired structure is a matter of trial and error. The flow rate of the injection solution is controlled by a programmable syringe pump and this can also be varied between experiments to simulate di...

Access restricted. Please log in or start a trial to view this content.

Materiały

Name	Company	Catalog Number	Comments
Syringe Pump	Fisher	14-831-3	Dual or multiple channel, depending on desired number of simultaneous experiments
Ferrous chloride tetrahydrate	Fisher	I90500	Ferrous Chloride Tetrahydrate (Crystalline/Certified)
Ferric chloride hexahydrate	Fisher	I88-100	Ferric Chloride Hexahydrate (Lumps/Certified ACS)
Sodium hydroxide	Sigma-Aldrich	S5881	reagent grade, ≥98%, pellets (anhydrous)
Sodium sulfide nonahydrate	Fisher	S425212	Sodium Sulfide Nonahydrate (Crystalline/Certified ACS). Store at -20 °C. Only open in a glove box or fume hood. Releases toxic H2S gas; all sulfide-containing solutions must be kept in a glove box or fume hood.
Potassium pyrophosphate	Sigma-Aldrich	322431	97%
L-Alanine	Sigma-Aldrich	A7627
Syringes (10 cc)	Fisher	14-823-16E	BD™ Syringe with Luer-Lok Tips (Without Needle)
Syringe needles (16 gauge)	Fisher	14-826-18B	BD™ General Use and PrecisionGlide Hypodermic Needles, 16 G x 1.5 in. (38 mm)
Tubing	Cole Parmer	EW-06407-71	Tygon Lab Tubing, Non-DEHP, 1/16" ID x 1/8" OD
Aluminum seals	Fisher	0337523C	Thermo Scientific™ National™ Headspace 20 mm Crimp Seals
Gray butyl stoppers	Fisher	0337522AA	Thermo Scientific™ National™ 20 mm Septa for Headspace Vials
Serum bottles	Sigma-Aldrich	33110-U	Vials, crimp top, serum bottle, size 100 ml, clear glass, O.D. × H 51.7 mm × 94.5 mm. For these experiments, the bottom of the serum bottle should be cut off.
Pipette tips	VWR	53511-682	pipette tips 0.5-10 μl
Wire	McMaster-Carr	8073K661	Solid Single-Conductor Wire, UL 1007/1569, 20 AWG, 300 VAC