Kensei Kobayashi’s flow-reactor, the SCWFR, part 2

The SCWFR at Kensei Kobayashi's lab at Yokohama National University
The SCWFR at Kensei Kobayashi’s lab at Yokohama National University

Kensei Kobayashi’s group with the help of Takeo Kanaeko (a brilliant man) came up with another design where they named it Super Critical Water Flow Reactor (SCWFR) utilizing an infrared (IR) gold image furnace (Figure above). This enables the fluid in this system to heat up to 400 °C (or any desired temperature) within seconds (without pre-heating). This one specification is important as most autoclave and other flow reactors utilized an electrical heater which takes time to heat up to the desired temperature. This is undesirable as this “pre-heating” is also making the initial chemicals to react (or altered) to lower and rising temperature, which is not very realistic to a real vent. In other case, this is one of the slightly more realistic simulators i have worked to date.

Kobayashi’s ex-student, Nazrul Islam performed the survivability experiment using several amino acids (aspartic acid, threonine, serine, sarcosine, glutamic acid, α-aminobutyric acid, β- alanine, γ-aminobutyric acid, 5-aminovaleric acid and 6- aminohexanoic acid), 10 mM each) by heating them up at four different temperatures (250, 300, 350 and 400°C ) at 25 MPa for 2 min (that’s pretty fast). They showed that amino acids tend to show better recovery when it is hydrolyzed, suggesting some kind of aggregation and/or condensation is happening within the system. Additionally they also showed oligomerization of glycine (100mM) with the same temperature. At 400°C, no oligomerization was found. However, at 200-350°C, diketopiperazine, diglycine, triglycine and tetraglycine were formed. It was suggested that the glycine reactions in a supercritical fluid (300°C to 400°C) are quite different from those in the liquid phase (at 200–350°C). There were many other peaks of unknown compounds in all of the chromatograms of the unhydrolyzed products. Most of those peaks disappeared after acid hydrolysis. This particular findings however remains a mystery.

Another of Kensei’s sudent, Hironari Kurihara, used the SCWFR to test the stability of complex compounds produced from simulated primitive atmosphere (similar to Miller and Urey’s work). He then tested these compounds to assess the stability of some known amino acids. The complex-combined amino acids (from the primitive atmosphere simulation) preserved more amino acids (including amino acid precursors that give amino acids after acid hydrolysis) than free amino acids after heating in simulated SHS environments. In other words, these complex chemicals which could come from atmosphere (or space) could be better preserved if they are come into contact with the hot hydrothermal system which could have existed during the prebiotic-earth setting. This also hints that “clean or perfect” chemicals like amino acids used in many kinds of experiments are not necessarily showing the right picture when comes to chemical evolution on early earth. More on this in the future.

Many critics of hydrothermal vent simulators often argues that, simulators mentioned on this post and previous ones have a rather short flow-rate and short flow-length (showing low residence time, meaning how long the sample stays in the system). Critics usually cite studies that refer to residence time of fluids in axial hydrothermal environments range from years to decades, while those in lower temperature off-axis diffuse flow systems may be on the order of thousands of years. The problem with these studies about residential axial time of fluids is that, they’re are based on models and calculation only, no work has been done so far in regards to real time sampling or measuring the length of vent system (although, there are work in measuring the velocity of fluid emitting from a vent which looks promising for future measuring). Despite this limitation, i believe the future of simulators will be done using computer simulators, where experimental data from physical simulators (like the ones mentioned in this blog) could be used as parameters. The biggest advantage in this, is that, we do experiments on a larger scale which is could make the origin of life more complicated, interesting and not so straightforward, which I believe is a good problem to have in the future.

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The flow-reactor, a better hydrothermal vent simulator, part 1

flow-reactor (HCFR) build by Ei-ichi Imai at Nagaoka Institute of Technology.
flow-reactor (HCFR) build by Ei-ichi Imai at Nagaoka Institute of Technology.

In the previous post, i wrote about a conventional kind of submarine hydrothermal simulator (SHS) called the autoclave. In today’s post i will talk about the flow kind of reactor which is more realistic compared to the autoclave. Jack Corliss, the man who discovered a real SHS, came up with the notion that a flow-type reactor is essential for new origin of life experiments, as he argued that the quenching effect of the cold seawater is important to hold the short-lived intermediates (hydrogen cyanide, sugars and etc) or as he calls it “quasi-species” in prebiotic synthesis of bio-molecules. This essentially means that a flow kind of simulators are required to better represent a real SHS to  produce more reliable results than the autoclave.

Initial Schematics of Jack Corliss in his paper
Initial Schematics of Jack Corliss in his paper

Co-incidentally, Koichiro Matsuno, designed a principle idea of a flow reactor which could reflow the fluids repeatedly in heat and cold (i will name here-forth as hydrothermal circulation flow-reactor (HCFR) for convenience). Ei-Ichi Imai, Koichiro Matsuno and co-workers went on to build  (image above, schematics diagram below) the reactor and experimented using many bio-molecules.

Their first paper which made it to Nature (big thing for scientist), reported that oligomerization of glycine could occur at 200-250°C, where a monomer glycine solution was circulating for 2 hours or so. Oligomers up to Hexaglycine was reported when metal (Cu2+) and controlled pH was introduced. Without them, only oligomers up to triglycine could be obtained. They also tried the same experiment using  glycine and alanine. They observed oligomerzation products such as

Schematic diagram of the HCFR system from Nagaoka Institute of Technology
Schematic diagram of the HCFR system from Nagaoka Institute of Technology

diketopiperaxine, gly-ala, ala-gly, gly-ala-ala, ala-ala, ala-ala-ala and ala-ala-ala- ala was created when the solution was heated with similar conditions. What i described abover is a big thing in origin of life studies. This land-mark discovery, was the first to show that oligomerization (a shorter version of polymerization) could occur using basic amino acid (gly), without the help of DNA and RNA molecules which are crucial in making proteins in life.

In their other works, oligomerization of nucleotide up to trimers (basic monomer of DNA) was also observed when 20mM of adenosine monophosphate was used with 1mM ZnCl2 were reflowed at 110°C.

Despite the findings made by HCFR, the system is rather hypothetical, since we know that real live vents don’t really circulate in that manner (shown below) however, this kind of reactor is useful when it comes to monitor chemical changes in the system. In my next post, i will review another kind of flow-reactor in Japan which i have also worked on in the past.

HCFR mimicking a circulatory system in a real life vent.
HCFR mimicking a hypothetical circulatory system in a real life vent.

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Origin of life – a brief history

The study of astrobiology involves 3 things, finding the origin of life, finding aliens and also the future of life. The latter is fully theoretical and often described as the one for the future. When it comes to finding aliens (FA), we are looking for more simpler life forms (bacteria etc), the complex ones are mostly being investigated by the UFO junkies. I will touch on FA in the future. My focus today will be on origin of life (OL).

Viewpoints from all over the world has shaped the philosophy and early sciences in regards to the OL. In 1778, Buffon, a french naturalist was one of the first to postulate the origin of life theory, he claimed that during the early stages, with colder temperature (probably referring to the ice age), organic molecules could produce spontaneous generations, thus producing each species as we know them now. 80 years on, Charles Darwin wrote a letter to Joseph Hooker, a botanist describing a primordial condition on earth:

 It is often said that all the conditions for the first production of a living organism are now present, which could have been present. But if (and oh what a big if) we could conceive in some warm little pond with all sort of ammonia and phosphoric salts,—light, heat, electricity, present, that a protein compound was chemically formed, ready to undergo still more complex changes, at the present day such matter would be instantly devoured, or absorbed, which would not have been the case before living creatures were formed.

Darwin was referring to the OL. He did not define or even hinted about how the origin of life might have been in his famous book. Since his work in “origin of species” involved the history of life, he must have thought that there must have been a starting point. However, OL questions were considered buried once Louis Pasteur concluded that life came from preexisting life-forms. He stood by his words that there is a difference between non-life and life, hence life would not have occurred by inert molecules. In 1917, a young Russian, Aleksandr Oparin begin thinking; How did life first started. He wrote his thoughts in his book The origin of life; published in 1924. During the same time JBS Haldane, a British born geneticist independently publish an essay indicating the similar ideas to those of Oparin’s. Hence, the Oparin-Haldane hypothesis was introduced. Oparin suggested that organic chemicals could have went through a series of reactions which would then lead to more complex aggregates. The aggregates then form a colloidal mixture in watery environment. He calls them ‘coacervates’. Coacervates would be able to perform simple metabolic functions and with evolution become the first life form. Haldane posed a similar idea where the primordial sea would serve as a chemical reservoir powered by the sun. He also added that the group of chemicals was enclosed in a lipid membrane system, thus developing to become the first living cells. He coined the term ‘prebiotic soup’, and this became the expression of the Oparin-Haldane view of the origin of life.

In the early 1950s, Harold Urey, already a nobel price winner for his discovery of heavy water simulated a chemical model of primitive atmosphere. This was done with his then graduate student, Stanley Miller. They put up a mixture of simple gas to an electrical discharge, any products from this will accumulate in the flask below the apparatus. They left the experiment to run for a week. The products of the experiment contained organic chemicals including several amino acid (monomer of proteins). This results were indeed ground breaking and became an instant sensation among fellow scientist. The detection of amino acid shows that natural laws allows them to be synthesized under prebiotic condition. Till date many modified version of this Miller-Urey experiment has been conducted which further enhance our knowledge of creating amino acids from simple gas or molecules. Miller-Urey experiment started the bandwagon, on what kind of life’s chemicals can be created prebioticaly. John Oro showed that the highly reactive hydrogen cyanide under alkaline conditions will polymerize to become adenine, a nucleobase essential to DNA. Lastly in 1978, Dave Deamer and co-workers managed to produce vesicle from fatty acids under certain prebiotic condition. The vesicle here mimics a simple membraneous vesicles. He then used fatty acid extracted from Murchison meteorite (carbonaceous chondrites) to produce vesicle with prebiotic conditions. This indicates that fatty acid formed in space had/has the capacity to form vesicles which can encapsulate DNA or proteins to possibly form the Proto-cell.

The discoveries mentioned above are landmark discoveries which gave scientist the hope that we can get closer to the origin. The experiments (and many more) indicates that under prebiotic condition we can produce life’s chemicals. When it was discovered that Europa could have an ocean below its ice crust; and Mars used to have seas, scientist became excited because if Primitive Earth can yield life’s chemicals towards living beings, then the same could have/had happened in Mars and Europa respectively.

What i have mentioned above is simply a brief history of OL, many experiments have been conducted ever since. Its worth to mention that there are many limitation and unsolved riddles when comes to this study. For example, although we can produce amino acid prebioticaly, there isn’t a prebiotic way where we can polymerize the amino acids to proteins. Also on close examination, life is very selective; only left handed amino acids are utilized to perform life’s activities. But in prebiotic experiments (e.g Miller-Urey), a racemic mixture is always produced, in other words 50% right handed and 50% left handed amino acids. There are many hypothesis that can explain on why these phenomena known as homochirality happens, but there are no explanations on HOW this must have happened 3.5 billion years ago. If we look further into the formation of genetic materials, it is known that DNA denature under prebiotic temperatures (200 C to 400 C). So how did the DNA molecules formed when the earth was hot as hell?. This is only the the tip of the iceberg. Till then this is the story we have.

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