This research was carried out at the Jet Propulsion Laboratory, California Institute of Technology, under a contract with the National Aeronautics and Space Administration. We acknowledge the support by the NASA Astrobiology Institute (Icy Worlds). L.M.B. is supported by the NAI through the NASA Postdoctoral Program, administered by Oak Ridge Associated Universities through a contract with NASA. J.E.N. was supported through a US Department of Education PR/Award #: P031C110019 administered through Citrus College. We acknowledge useful discussions with members of the NAI Thermodynamics, Disequilibrium, and Evolution Focus Group and the Blue Marble Space Institute of Science.

1. Safety Considerations
<ol>
	<li>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.</li>
	<li>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.
	<ol>
		<li>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&#8226;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.</li>
	</ol>
	</li>
</ol>
2. Setup for Injection Experiments
<ol>
	<li>Create glass &#8220;injection vials&#8221; 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.</li>
	<li>Prepare the injection vials (Figure 1).
	<ol>
		<li>Collect a 20 mm septum, 20 mm aluminum crimp seal, and a 0.5-10 &#181;l 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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Affix 1/16&#8221; 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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Set up electrodes for measuring membrane potential across the wall of the chemical gardens. Always use the same convention for which lead is &#8220;inside&#8221; and which is &#8220;outside&#8221; of the chemical gardens.
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Tape or otherwise secure the wires so that they cannot move inside the injection vial during the experiment (Figure 2).</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Set up N2 gas lines that will each feed into one of the injection vials.
	<ol>
		<li>Split the gas feed from a N2 source into several tubes, so that there is one N2 feed for each injection vial.</li>
		<li>Place each N2 tube so that it feeds into the headspace of one of the injection vials.</li>
	</ol>
	</li>
</ol>
3. Preparation of Solutions for Chemical Garden Growth
<ol>
	<li>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).
	<ol>
		<li>Create anoxic solutions by first bubbling the ddH2O with N2 gas for ~15 min per 100 ml.</li>
		<li>Weigh out and add the FeCl2&#8226;4H2O and FeCl3&#8226;6H2O, stirring gently to dissolve (not vigorously so as to not introduce oxygen).</li>
		<li>After reagents are dissolved, immediately resume light bubbling of the Fe2+/Fe3+ solution with N2 gas while injections are prepared.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Prepare 20 ml of the secondary injection solution (sodium sulfide &#8212; 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.</li>
	<li>Refill the ddH2O syringes from Step 2.2.5; these will be used to flush the injection tube.</li>
</ol>
4. Starting the Primary Injection
<ol>
	<li>Use desired data logger for membrane potential measurements; measure each experiment&#8217;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).</li>
	<li>Secure the primary injection syringes on the programmable syringe pump in the fume hood.</li>
	<li>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).</li>
	<li>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.</li>
	<li>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.</li>
	<li>Pour 100 ml of the Fe2+/Fe3+ reservoir solution into each vial.</li>
	<li>Adjust the flow of the N2 gas lines as desired to keep the experiment anoxic for the duration of the injections.</li>
	<li>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).</li>
	<li>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.)</li>
	<li>Start the injection, and start recording of membrane potential.</li>
</ol>
5. Starting the secondary injection:
<ol>
	<li>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).</li>
	<li>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.</li>
	<li>Secure the secondary injection sulfide syringes to the syringe pump, and repeat Steps 4.3 and 4.4.</li>
	<li>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.</li>
	<li>When the transfer is complete, carefully secure the secondary syringes to the syringe pump.</li>
	<li>Re-program the syringe pump to inject at 2 ml per hour, and hit start to continue the injection with the new injection solution.</li>
	<li>Safely dispose of the primary injection syringes.</li>
</ol>
6. Ending the Experiment
<ol>
	<li>First stop the syringe pump, then stop recording of the membrane potential and save the data.</li>
	<li>Turn off the N2 flow and remove the lines and the Parafilm from the injection vessels.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Rinse out the glass experiment vial and soak it in a 1 M HCl acid bath O/N. (CAUTION &#8212; 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.)</li>
</ol>

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<li>Trolard, F., Bourrié, G. Fougerite a natural layered double hydroxide in gley soil: habitus, structure, and some properties. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aTrolard%2C+F+%22Clay+Minerals+in+Nature+-+Their+Characterization%2C+Modification+and+Application%22">Clay Minerals in Nature - Their Characterization, Modification and Application</a>. , InTech. (2012).</li>
<li>van Oss, C. J. Specifically Impermeable Precipitate Membranes. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3avan+Oss%2C+CJ+%22Surface+and+Colloid+Science%22">Surface and Colloid Science</a>. , Springer. US. 115-144 (1984).</li>
<li>Baaske, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extreme+accumulation+of+nucleotides+in+simulated+hydrothermal+pore+systems%5BTitle%5D)+AND+%22P.+Natl.+Acad.+Sci.+USA%22%5BJournal%5D)">Extreme accumulation of nucleotides in simulated hydrothermal pore systems.</a> P. Natl. Acad. Sci. USA. 104, 9346-9351 (2007).</li>
</ol>

The authors declare that they have no competing financial interests.

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...

&#8220;Chemical gardens&#8221; are self-assembling inorganic precipitates developed where two fluids of contrasting chemistries interact1,2. 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 &#8220;chimney&#8221; 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 FeIII, Ni2+, Mn2+, NO3-, and NO2-. 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 &#8212; such as early Mars, Jupiter&#8217;s moon Europa, or Saturn&#8217;s moon Enceladus &#8212; it is possible that water/rock chemistry could generate alkaline vent environments capable of driving prebiotic chemistry or even providing habitable niches for extant life5,9-11.
The classic chemical garden experiment involves a seed crystal of a metal salt, e.g. ferrous chloride tetrahydrate FeCl2&#8226;4H2O, submerged in a solution containing reactive anions, e.g. sodium silicate or &#8220;water glass&#8221;. 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 potential12-14. Chemical garden structures are complex, exhibiting compositional gradients from interior to exterior13,15-19, 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 materials20,21.
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 species20,23.
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 years24,25.

<table><tbody><tr><td>Syringe Pump</td><td>Fisher</td><td>14-831-3</td><td>Dual or multiple channel, depending on desired number of simultaneous experiments</td></tr><tr><td>Ferrous chloride tetrahydrate</td><td>Fisher</td><td>I90500</td><td>Ferrous Chloride Tetrahydrate (Crystalline/Certified)</td></tr><tr><td>Ferric chloride hexahydrate</td><td>Fisher</td><td>I88-100</td><td>Ferric&amp;nbsp;Chloride&amp;nbsp;Hexahydrate&amp;nbsp;(Lumps/Certified ACS)</td></tr><tr><td>Sodium hydroxide</td><td>Sigma-Aldrich</td><td>S5881</td><td>reagent grade, &amp;ge;98%, pellets (anhydrous)</td></tr><tr><td>Sodium sulfide nonahydrate</td><td>Fisher</td><td>S425212</td><td>Sodium Sulfide Nonahydrate (Crystalline/Certified ACS). Store at -20&amp;nbsp;&amp;#176;C. Only open in a glove box or fume hood. Releases toxic H&lt;sub&gt;2&lt;/sub&gt;S gas; all sulfide-containing solutions must be kept in a glove box or fume hood.</td></tr><tr><td>Potassium pyrophosphate</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/aldrich/322431?lang=en&amp;amp;region=US&quot;&gt;322431&lt;/a&gt;</td><td>97%</td></tr><tr><td>L-Alanine</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sigma/a7627?lang=en&amp;amp;region=US&quot;&gt;A7627&lt;/a&gt;</td><td></td></tr><tr><td>Syringes (10&amp;nbsp;cc)</td><td>Fisher</td><td>14-823-16E</td><td>BD&amp;trade; Syringe with Luer-Lok Tips (Without Needle)</td></tr><tr><td>Syringe needles (16&amp;nbsp;gauge)</td><td>Fisher</td><td>14-826-18B</td><td>BD&amp;trade; General Use and PrecisionGlide Hypodermic Needles, 16&amp;nbsp;G x 1.5 in. (38&amp;nbsp;mm)</td></tr><tr><td>Tubing</td><td>Cole Parmer</td><td>EW-06407-71</td><td>Tygon Lab Tubing, Non-DEHP, 1/16&quot; ID x 1/8&quot; OD</td></tr><tr><td>Aluminum seals</td><td>Fisher</td><td>0337523C</td><td>Thermo Scientific&amp;trade; National&amp;trade; Headspace 20&amp;nbsp;mm Crimp Seals</td></tr><tr><td>Gray butyl stoppers</td><td>Fisher</td><td>0337522AA</td><td>Thermo Scientific&amp;trade; National&amp;trade; 20&amp;nbsp;mm Septa for Headspace Vials</td></tr><tr><td>Serum bottles</td><td>Sigma-Aldrich</td><td>33110-U</td><td>Vials, crimp top, serum bottle, size 100 ml, clear glass, O.D. &amp;times; H 51.7 mm &amp;times; 94.5 mm. For these experiments, the bottom of the serum bottle should be cut off.</td></tr><tr><td>Pipette tips</td><td>VWR</td><td>53511-682</td><td>pipette tips 0.5-10 &amp;#956;l</td></tr><tr><td>Wire</td><td>McMaster-Carr</td><td>8073K661</td><td>Solid Single-Conductor Wire, UL 1007/1569, 20 AWG, 300 VAC</td></tr></tbody></table>

chemical gardens as flow-through reactors simulating natural hydrothermal systems

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 &#8220;hydrothermal&#8221; injections.

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...

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Chemical Gardens as Flow-through Reactors Simulating Natural Hydrothermal Systems

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

Here we report experimental simulations of hydrothermal chimney growth using injection chemical garden methods. The versatility of this type of experiment ...

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Research

JoVE Journal

Chemistry

14.5K Views.  California Institute of Technology. We describe chemical garden formation via injection experiments that allow for laboratory simulations of natural chemical garden systems that form at submarine hydrothermal vents.

Video: Chemical Gardens as Flow-through Reactors Simulating Natural Hydrothermal Systems

Millifluidics for Chemical Synthesis and Time-resolved Mechanistic Studies

Procedures utilizing millifluidic devices for chemical synthesis and time-resolved mechanistic studies are described by taking three examples. In the first, synthesis of ultra-small copper nanoclusters is described. The second example provides their utility for investigating time resolved kinetics of chemical reactions by analyzing gold nanoparticle formation using in situ X-ray absorption spectroscopy. The final example demonstrates continuous flow catalysis of reactions inside millifluidic channel coated with nanostructured catalyst.

Millifluidic devices are utilized for controlled synthesis of nanomaterials, time-resolved analysis of reaction mechanisms and continuous flow catalysis.

Procedures utilizing millifluidic devices for chemical synthesis and time-resolved mechanistic studies are described by taking three examples. In the ...

Bioengineering

Continuous Flow Chemistry: Reaction of Diphenyldiazomethane with p-Nitrobenzoic Acid

Continuous flow technology has been identified as instrumental for its environmental and economic advantages leveraging superior mixing, heat transfer and cost savings through the &#34;scaling out&#34; strategy as opposed to the traditional &#34;scaling up&#34;. Herein, we report the reaction of diphenyldiazomethane with p-nitrobenzoic acid in both batch and flow modes. To effectively transfer the reaction from batch to flow mode, it is essential to first conduct the reaction in batch. As a consequence, the reaction of diphenyldiazomethane was first studied in batch as a function of temperature, reaction time, and concentration to obtain kinetic information and process parameters. The glass flow reactor set-up is described and combines two types of reaction modules with &#34;mixing&#34; and &#34;linear&#34; microstructures. Finally, the reaction of diphenyldiazomethane with p-nitrobenzoic acid was successfully conducted in the flow reactor, with up to 95% conversion of the diphenyldiazomethane in 11 min. This proof of concept reaction aims to provide insight for scientists to consider flow technology&#39;s competitiveness, sustainability, and versatility in their research.

Continuous Flow Chemistry: Reaction of Diphenyldiazomethane with p-Nitrobenzoic Acid

Flow chemistry carries environmental and economic advantages by leveraging superior mixing, heat transfer and cost benefits. Herein, we provide a blueprint to transfer chemical processes from batch to flow mode. The reaction of diphenyldiazomethane (DDM) with p-nitrobenzoic acid, conducted in batch and flow, was chosen for proof of concept.

Continuous flow technology has been identified as instrumental for its environmental and economic advantages leveraging superior mixing, heat transfer and ...

Utilization of Stop-flow Micro-tubing Reactors for the Development of Organic Transformations

A new reaction screening technology for organic synthesis was recently demonstrated by combining elements from both continuous micro-flow and conventional batch reactors, coined stop-flow micro-tubing (SFMT) reactors. In SFMT, chemical reactions that require high pressure can be screened in parallel through a safer and convenient way. Cross-contamination, which is a common problem in reaction screening for continuous flow reactors, is avoided in SFMT. Moreover, the commercially available light-permeable micro-tubing can be incorporated into SFMT, serving as an excellent choice for light-mediated reactions due to a more effective uniform light exposure, compared to batch reactors. Overall, the SFMT reactor system is similar to continuous flow reactors and more superior than batch reactors for reactions that incorporate gas reagents and/or require light-illumination, which enables a simple but highly efficient reaction screening system. Furthermore, any successfully developed reaction in the SFMT reactor system can be conveniently translated to continuous-flow synthesis for large scale production.

A protocol for organic reaction screening using stop-flow micro-tubing (SFMT) reactors employing gaseous reactants and/or visible-light mediated reactions is presented.

A new reaction screening technology for organic synthesis was recently demonstrated by combining elements from both continuous micro-flow and conventional ...

Microalgae Cultivation and Biomass Quantification in a Bench-Scale Photobioreactor with Corrosive Flue Gases

Photobioreactors are illuminated cultivation systems for experiments on phototrophic microorganisms. These systems provide a sterile environment for microalgal cultivation with temperature, pH, and gas composition and flow rate control. At bench-scale, photobioreactors are advantageous to researchers studying microalgal properties, productivity, and growth optimization. At industrial scales, photobioreactors can maintain product purity and improve production efficiency. The video describes the preparation and use of a bench-scale photobioreactor for microalgal cultivation, including the safe use of corrosive gas inputs, and details relevant biomass measurements and biomass productivity calculations. Specifically, the video illustrates microalgal culture storage and preparation for inoculation, photobioreactor assembly and sterilization, biomass concentration measurements, and a logistic model for microalgal biomass productivity with rate calculations including maximum and overall biomass productivities. Additionally, since there is growing interest in experiments to cultivate microalgae using simulated or real waste gas emissions, the video will cover the photobioreactor equipment adaptations necessary to work with corrosive gases and discuss safe sampling in such scenarios.

Bench-scale, axenic cultivation facilitates microalgal characterization and productivity optimization before subsequent process scale-up. Photobioreactors provide the necessary control for reliable and reproducible microalgal experiments and can be adapted to safely cultivate microalgae with the corrosive gases (CO2, SO2, NO2) from municipal or industrial combustion emissions.

Photobioreactors are illuminated cultivation systems for experiments on phototrophic microorganisms. These systems provide a sterile environment for ...

Environment

Coupling Carbon Capture from a Power Plant with Semi-automated Open Raceway Ponds for Microalgae Cultivation

In the United States, 35% of the total carbon dioxide (CO2) emissions come from the electrical power industry, of which 30% represent natural gas electricity generation. Microalgae can biofix CO2 10 to 15 times faster than plants and convert algal biomass to products of interest, such as biofuels. Thus, this study presents a protocol that demonstrates the potential synergies of microalgae cultivation with a natural gas power plant situated in the southwestern United States in a hot semi-arid climate. State-of-the-art technologies are used to enhance carbon capture and utilization via the green algal species Chlorella sorokiniana, which can be further processed into biofuel. We describe a protocol involving a semi-automated open raceway pond and discuss the results of its performance when it was tested at the Tucson Electric Power plant, in Tucson, Arizona. Flue gas was used as the main carbon source to control pH, and Chlorella sorokiniana was cultivated. An optimized medium was used to grow the algae. The amount of CO2 added to the system as a function of time was closely monitored. Additionally, other physicochemical factors affecting algal growth rate, biomass productivity, and carbon fixation were monitored, including optical density, dissolved oxygen (DO), electroconductivity (EC), and air and pond temperatures. The results indicate that a microalgae yield of up to 0.385 g/L ash-free dry weight is attainable, with a lipid content of 24%. Leveraging synergistic opportunities between CO2 emitters and algal farmers can provide the resources required to increase carbon capture while supporting the sustainable production of algal biofuels and bioproducts.

A protocol is described to utilize the carbon dioxide in natural gas power plant flue gas to cultivate microalgae in open raceway ponds. Flue gas injection is controlled with a pH sensor, and microalgae growth is monitored with real time measurements of optical density.

In the United States, 35% of the total carbon dioxide (CO2) emissions come from the electrical power industry, of which 30% represent natural gas ...

Simulation of Early Earth Hydrothermal Chimneys in a Thermal Gradient Environment

Deep sea hydrothermal vents are self-organizing precipitates generated from geochemical disequilibria and have been proposed as a possible setting for the emergence of life. The growth of hydrothermal chimneys in a thermal gradient environment within an early Earth vent system was successfully simulated by using different hydrothermal simulants, such as sodium sulfide, which were injected into an early Earth ocean simulant containing dissolved ferrous iron. Moreover, an apparatus was developed to sufficiently cool the ocean simulant to near 0 &#176;C in a condenser vessel immersed in a cold water bath while injecting a sulfide solution at hot to room temperatures, effectively creating an artificial chimney structure in a temperature gradient environment over a period of a few hours. Such experiments with different chemistries and variable temperature&#160;gradients resulted in a variety of morphologies in the chimney structure. The use of ocean and hydrothermal fluid simulants at room temperature resulted in vertical chimneys, whereas the combination of a hot hydrothermal fluid and cold ocean simulant inhibited the formation of robust chimney structures. The customizable 3D printed condenser created for this study acts as a jacketed reaction vessel that can be easily modified and used by different researchers. It will allow the careful control of injection rate and chemical composition of vent and ocean simulants, which should help accurately simulate prebiotic reactions in chimney systems with thermal gradients similar to those of natural systems.

The goal of this protocol is to form simulated hydrothermal chimneys via chemical garden injection experiments and introduce a thermal gradient across the inorganic precipitate membrane, using a 3D printable condenser that can be reproduced for educational purposes.

Deep sea hydrothermal vents are self-organizing precipitates generated from geochemical disequilibria and have been proposed as a possible setting for the ...

A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past studies have evaluated the performance of the reactor for the removal of COD1 and nitrogen species2-4 by heterotrophic and chemoautotrophic bacteria, respectively. The HDBR design eliminates the requirement for external flocculation/sedimentation processes while still yielding effluent containing low suspended solids. In this study, the HDBR is applied as a photobioreactor (PBR) in order to characterize the nitrogen removal characteristics of an algae-based photosynthetic microbial community. As previously reported for this HDBR design, a stable biomass zone was established with a clear delineation between the biologically active portion of the reactor and the recycling reactor fluid, which resulted in a low suspended solid effluent. The algal community in the HDBR was observed to remove 18.4% of total nitrogen species in the influent. Varying NH4+ and NO3- concentrations in the feed did not have an effect on NH4+ removal (n=44, p=0.993 and n=44, p=0.610 respectively) while NH4+ feed concentration was found to be negatively related with NO3- removal (n=44, p=0.000) and NO3- feed concentration was found to be positively correlated with NO3- removal (n=44, p=0.000). Consistent removal of NH4+, combined with the accumulation of oxidized nitrogen species at high NH4+ fluxes indicates the presence of ammonia- and nitrite-oxidizing bacteria within the microbial community.

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Here, the HDBR is successfully applied in a photobioreactor (PBR) configuration for the study of nitrogen metabolism by a mixed high density algal community.

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past ...

Using Flexible Gold-Titanium Reaction Cells to Simulate Pressure-Dependent Microbial Activity in the Context of Subsurface Biomining

Laboratory studies investigating subsurface microbial processes, such as metal leaching in deep ore deposits (biomining), share common and challenging obstacles, including the special environmental conditions that need to be replicated, e.g., high pressure and in some cases acidic solutions. The former requires an experimental setup suitable for pressurization up to 100 bar, while the latter demands a fluid container with high chemical resistance against corrosion and unwanted chemical reactions with the container wall. To meet these conditions for an application in the field of in situ biomining, a special flexible gold-titanium reaction cell inside a rocking high-pressure reactor was used in this study. The described system allowed simulation of in situ biomining through sulfur-driven microbial iron reduction in an anoxic, pressure-controlled, highly chemically inert experimental environment. The flexible gold-titanium reaction cell can accommodate up to 100 mL of sample solution, which can be sampled at any given time point while the system maintains the desired pressure. Experiments can be performed on timescales ranging from hours to months. Assembling the high-pressure reactor system is fairly time consuming. Nevertheless, when complex and challenging (microbiological) processes occurring in the earth&#39;s deep subsurface in chemically aggressive fluids have to be investigated in the laboratory, the advantages of this system outweigh the disadvantages. The results found that even at high pressure the microbial consortium is active, but at significantly lower metabolic rates.

This protocol describes microbial experiments under elevated pressures to study in situ biomining processes. The experimental approach employs a rocking high-pressure reactor equipped with a gold-titanium reaction cell containing a microbial culture in an acidic, iron-rich medium.

Laboratory studies investigating subsurface microbial processes, such as metal leaching in deep ore deposits (biomining), share common and challenging ...

Engineering

Mesocosm-Scale Constructed Wetland Design for Wastewater Treatment

Oil sands process-affected water (OSPW), a by-product of bitumen extraction through surface mining in Alberta, Canada, contains various constituents of concern, including naphthenic acid fraction compounds (NAFCs). These organic compounds are particularly worrisome due to their toxicity and persistence in the environment. Constructed wetland treatment systems (CWTS) use plants and their associated microbes to attenuate contaminants in wastewater. Field-scale CWTS have been presented as a potential large-scale treatment option for OSPW, specifically for degrading NAFCs. To optimize the use of CWTS for large-scale treatment of NAFCs in OSPW, it is essential to deepen our understanding of various design parameters and explore ways to enhance efficacy.
Mesocosm-scale experiments serve as a valuable intermediary, bridging the gap between complex field trials and controlled laboratory settings. Mesocosms provide a controlled, replicable environment to study the effects of various parameters such as substrate, plant species, temperature, and retention time while incorporating ecological complexities in their design. Published and previous work has shown that this method is successful in evaluating the impacts of different parameters on the efficacy of CWTS to attenuate NAFCs in OSPW. This protocol outlines the design and setup of a surface flow wetland mesocosm, along with the experimental approach for treating NAFCs in OSPW. This method can be adapted to treat other wastewaters across diverse geographical locations.

Constructed wetland treatment systems have been used for decades to treat wastewater, but their application to treat oil sands process-affected waters is relatively new. To explore this potential, a surface flow mesocosm design and experimental methods are outlined. This approach aims to enhance our understanding of key design parameters and improve treatment efficacy.

Oil sands process-affected water (OSPW), a by-product of bitumen extraction through surface mining in Alberta, Canada, contains various constituents of ...

Window on a Microworld: Simple Microfluidic Systems for Studying Microbial Transport in Porous Media

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in the environment, as well as directly for the transmission of pathogens to drinking water supplies. Natural porous media is composed of an intricate physical topology, varied surface chemistries, dynamic gradients of nutrients and electron acceptors, and a patchy distribution of microbes. These features vary substantially over a length scale of microns, making the results of macro-scale investigations of microbial transport difficult to interpret, and the validation of mechanistic models challenging. Here we demonstrate how simple microfluidic devices can be used to visualize microbial interactions with micro-structured habitats, to identify key processes influencing the observed phenomena, and to systematically validate predictive models. Simple, easy-to-use flow cells were constructed out of the transparent, biocompatible and oxygen-permeable material poly(dimethyl siloxane). Standard methods of photolithography were used to make micro-structured masters, and replica molding was used to cast micro-structured flow cells from the masters. The physical design of the flow cell chamber is adaptable to the experimental requirements: microchannels can vary from simple linear connections to complex topologies with feature sizes as small as 2 &mu;m. Our modular EcoChip flow cell array features dozens of identical chambers and flow control by a gravity-driven flow module. We demonstrate that through use of EcoChip devices, physical structures and pressure heads can be held constant or varied systematically while the influence of surface chemistry, fluid properties, or the characteristics of the microbial population is investigated. Through transport experiments using a non-pathogenic, green fluorescent protein-expressing Vibrio bacterial strain, we illustrate the importance of habitat structure, flow conditions, and inoculums size on fundamental transport phenomena, and with real-time particle-scale observations, demonstrate that microfluidics offer a compelling view of a hidden world.

Microfluidic devices can be used to visualize complex natural processes in real time and at the appropriate physical scales. We have developed a simple microfluidic device that mimics key features of natural porous media for studying growth and transport of bacteria in the subsurface.

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in ...

Chemical Gardens as Flow-through Reactors Simulating Natural Hydrothermal Systems

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