Does Enceladus’ alkaline ocean make it friendly to life?

Recent data sent back to us by the Cassini space probe as it samples the geyser water being shot into space at Saturn’s moon Enceladus, has determined that the moon’s subsurface ocean has a very high pH.  The pH is estimated to be around 11 or 12.  This would be considered extremely alkaline, but the team analyzing the data concludes that this might improve the odds of supporting life.  They point to the alkaline hydrothermal vents, such as The Lost City, on the ocean floor of earth, where warm alkaline fluids flow out into the cold salty deep.  There is some thought in the astrobiology community that life on earth may have originated in a similar alkaline vent environment 4.5 billion years ago.  The difference, however, is that on early earth the alkaline vent fluid was flowing into an acidic ocean, with a thin mineral wall separating the fluids and allowing a proton gradient to form.  It was this proton gradient that generated the energy necessary to transport electrons from molecule to molecule.  This is exactly what living organisms do to generate energy – they pump protons across a cell membrane, transport electrons to an ultimate electron acceptor (oxygen in our case), and use the proton gradient to generate ATP (the energy currency of the cell).  Cells do the biological equivalent of what the alkaline vents are doing geochemically.  For that reason I wonder if the high pH of Enceladus’ ocean really would support the origin of life since it doesn’t necessarily imply situation where a proton gradient would occur.

 

Reference:

How Friendly is Enceladus’ Ocean to Life?  Astrobiology magazine.  Feb. 4, 2016

Book Review: “The Vital Question”

By Rich Feldenberg:

On this episode of Darwin’s Kidneys – first of 2016- I’ll be reviewing a book by Nick Lane called, “The Vital Question: Energy, Evolution, and the Origins of Complex Life”. This book attempts to tackle some of the toughest questions in biology today, such as how, and in what environments, life originated, how the complex eukaryotic cell evolved, how the cellular mechanisms to generate energy echo back to the days before biology, and why sexual reproduction is the way it is based constraints placed on us by our energy generating systems -the mitochondria. It is a lot of territory to cover, but Dr. Lane does an amazing job of bringing all these seemingly diverse themes together, synthesizing them into a coherent narrative that flows as easily from one topic to the next, as electrons flow down the mitochondrial respiratory chain (a central subject of the book).

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For those of you, who like me, love the topic of biological origins, this book will keep you engaged, and I had trouble putting it down, as I waiting for the next amazing revelation to be exposed. The early part of the book describes the common thread between the most essential metabolic activities of all living cells on earth -whether they are bacteria, archaea, or complex eukaryotes – and the natural geochemical activity of Alkaline Hydrothermal Vents. All life generates its energy by using proton gradients to drive the production of ATP (the energy currency of the cell). In all cells today, special pumps have evolved to pump protons (hydrogen ions) across a membrane. This creates a proton gradient (more protons on one side of the membrane than the other) which will naturally lead to those protons tending to diffuse back across the membrane. Cells use this proton gradient to run the protein ATP-synthase, to generate ATP, just like running water can be used to turn a water wheel to do work at a mill. In order to get the proton, it has to be separated from its electron, and that is done through a series of oxidation-reduction (redox) reactions, where the electron is transferred from one compound to another with each subsequent compound having a greater affinity for the electron than the last compound. It ends with the electron being transferred to oxygen (O2), which has the most affinity for the electron, converting the oxygen to water. The compounds where the electron is being transferred, are the respiratory transport chain of proteins. It is also found in plants as part of their photosynthesis machinery.

mitochondria

electron transport

 

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This process mirrors a naturally occurring geological process found in Alkaline Hydrothermal Vents on the ocean floor. These vents are different from the “Black Smokers” that have been better popularized, as sites of chemosynthesis, where an ecology of organisms survive using the energy of the vent, and are not directly dependent on energy of the sun. The Alkaline Vents, on the other hand, are not quite so hot, but more importantly are composed of a matrix of mineral with thin walls that mimics a cell membrane. The vent fluid is more alkaline, with a pH of around 10, and the ocean water more acidic. It is thought that the ocean pH, 4.5 billion years ago might have been even more acidic that it is today with a pH of around 6. Since pH is a measure of the proton concentration, there is a natural proton gradient between vent fluid and ocean water separated by a thin mineral. The mineral also contains Iron-Sulfer complexes and other minerals that can act as redox centers, producing the electron transfer that we also still see today in our respiratory transport chain.

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Dr. Lane argues that this environment provides a very plausible explanation for how life originated and why all life uses the unusual proton gradient method to generate energy. His own research is, in part, using reactors to replicate the Alkaline Vent environment to study this theory further.
He goes on to discuss how life could then have evolved more effective cell membranes making wondering further from the vent location possible, as long as these simple organisms could begin to pump protons on their own, at this point. This movement into the new environment, and an existence independent of the Alkaline Vent, is where the split between bacteria and archaea probably occurred. He shows the evidence for this hypothesis.
A great deal of the rest of the book describes the evolution of the complex cell, by the synthesis of an archaea host cell, with a bacterial endosymbiont which went on to become the mitochondria. He also describes, in detail, the genetic evidence, as well as, that logical considerations, that suggest this occurred, it occurred only once, and how the other features of the complex cell -such as nuclear membrane developed.

Tree of life

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The book is beautifully written, but I will say some background in biology certain helps, but his writing is clear, entertaining, and well focused.
I just finished reading, “The Vital Question” this month, but it is now in my top 10 all time favorite science books. The last Nick Lane book I read was called, “Oxygen” and was equally good. It was also about the biochemistry of energy generation in organisms. I urge you to check out, “The Vital Question”, and let me know what you think.

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References:
1. The Vital Question, by Nick Lane

2. Nick Lane webpage.

3. Darwin’s Kidney Article on Molecular Fossils (EMMAs).

4. Article on the necessity of a new word, Mesign, to help differentiate between something purposefully designed and something that has the false appearance of design being evolved by natural selection.

Entropy as an engine of life’s origins

by Rich Feldenberg:

In our last Darwin’s Kidneys post we discussed the basic concept behind the second law of thermodynamics, which requires that entropy increase for every irreversible process. Entropy can be thought of as the amount of disorder in a system, so this law is essentially saying that there is an increase in the total amount of disorder that accompanies every physical process. We discussed why this law – which is thought to always hold true throughout time and space – does not prohibit the development of complex structure or the evolution of life, but it might also be true that the second law is a driving force behind the evolution of complexity in both living and non-living systems.

In this article I would like to continue our thermodynamic discussion, but introducing an interesting, although somewhat unproven and controversial offshoot of this scientific principle, which attempts to show that self organization of atoms and molecules is actually a consequence of second law dynamics. It’s founder and major proponent is a young physics professor at MIT, named Jeremy England. He has been attempting to show through a rigorous mathematical approach, that complexity arises naturally in physical systems as these systems move towards more efficient mechanisms to disperse energy – increase disorder in their surroundings. These systems become more efficient at increasing universal disorder, by becoming themselves more ordered. This work has potentially broad implications helping us understand how living systems might have arisen naturally from non-living systems, even before those systems were self-replicating and capable of Darwinian evolution.

The entropy of a closed system will always increase over time, but an open system allows an influx of energy so that the entropy of part of that system can decrease as the entropy of it’s surroundings increases. The geochemical environment of the early earth could be considered an open system because there was intense energy continuously entering into the system from the sun. Plants are extremely efficient at using that energetic sunlight to maximize the disorder of their surroundings. This is somewhat like looking at the problem upside down from our usual way of thinking. We normally think of plants evolving to use sunlight more effectively to become more complex, and as a natural consequence they create a larger entropy to the environment. England’s way of looking at the plant might be to say that second law demands that entropy will increase with time and the highly energetic sunlight will affect the system so that complexity will arise that will move towards maximum entropy generation. Those more effective entropy generators will necessarily be more complex systems, tending toward self-assembly and reproduction, and in some cases, eventually what we would recognize as living things. Living systems are very good at dissipating its energy.

 

thermodynamics of life

For these kind of processes to occur a system has to be out of thermodynamic equilibrium. At equilibrium there is no net energy transfer, but a system out of equilibrium has a net movement of energy – the influx of sunlight, for example. At some distant time in the future, the entropy of the entire universe will be high (the universe being a closed system), and at that point all areas of the universe will be in thermodynamic equilibrium, and complexity, organization, and life will cease to exist. Fortunately, it is likely to be a very long time before that fate befalls our universe.

England’s thermodynamic dissipative process might explain organized non-living structures we see everywhere in the world, from the formation of snowflakes and sand dunes, to planetary rings and spiral galaxies. These structures preferentially form to better disperse energy into more disordered and less usable forms – a consequence of thermodynamic’s second law. In this way, life itself is just one form of a more broad variation on this theme. Self organizing structures may have formed to raise entropy maximally, and in doing so lead to the first self-replicators. Once you have replicators, a Darwinian evolution by natural selection can take over to increase complexity further.

Not all researchers believe that Dr. England’s theory will pan out as a solution to the origin of life, but it seems that there are more than a few that have been impressed with the theory and its results so far. I have read two of England’s original journal articles, and unfortunately that math of the statistical mechanics was beyond me. From what other researchers have said, however, the equations used are valid, it is their interpretation for self assembly and origins of life, that is still unclear.

Professor England is himself and interesting individual. In his early 30s and approaching the origin of life field from a fresh perspective, England earned his PhD in physics at Stanford University in 2009, and is now an Assistant Professor of Physics at the Massachusetts Institute of Technology with his own research lab. In 2011 he was named as “one of the 30 under 30 rising stars in science”, by Forbes magazine. One thing that I found particularly fascinating is that although England is attempting to crack the tough nut of the origins of life, using sound science and mathematical modeling, he is a devout Orthodox Jew. He speaks somewhat to his faith and how he reconciles faith with his naturalistic scientific approach to answer this basic fundamental question, of interest to both science and religion, in his podcast interview that I linked to below. Faith and high level scientific inquiry may be a good topic for another time.

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I look forward to following Dr. England’s future work, and watching if others pick up on it and extend it further. If England is right, then far from The Second Law of Thermodynamics being a repressor of complexity, it may more accurately be a driving engine of the spontaneous production of organization and complex systems.

 

References:
1. “Statistical physics of self-replication”, Jeremy L. England; The Journal of Chemical Physics. 139, 121923 (2013).
2. “Dissipative adaptation in driven self-assembly”, J.L. England; Nat Nanotechnol. 10(11):919-23, Nov 4, 2015.
3. “The New Physics Theory of Life”. Quanta Magazine. January 22, 2014.
https://www.quantamagazine.org/20140122-a-new-physics-theory-of-life/
4. “Origins of Life: A Means to a Thermodynamically Favorable End?” Yale Scientific. July 1, 2014.
http://www.yalescientific.org/2014/07/origins-of-life-a-means-to-a-thermodynamically-favorable-end/
5. The 7th Avenue Project (Podcast). “Biophysicist Jeremy England: A New Theory of Life”. May 3, 2015.
http://7thavenueproject.com/post/118064180870/biophysicist-jeremy-england-new-theory-of-life
6. “How can we be so complex if the second law of thermodynamics is true?” Darwin’s Kidneys. Dec. 4, 2015.
http://darwinskidneys-science.com/2015/12/04/how-can-we-be-so-complex-if-the-second-law-of-thermodynamics-is-true/

 

You Must First Invent the Universe…

By Rich Feldenberg

This year Carl Sagan Day is being celebrated Saturday, November 14th. Sagan, who was born on November 9th, 1934 has been an inspiration to generations of scientists and science enthusiasts. Unfortunately, he passed away on December 20th, 1996 at the age of 62. Way too young, and certainly way too soon for a world that desperately needed his carefully measured dose of rationality, skepticism, and his poetic style of revealing the awe of the cosmos we inhabit together.

There are many great and inspiring Sagan quotes, or Saganisms as they’ve come to be known, but one of my favorites is, “If you want to make and apple pie from scratch, you must first invent the universe”. The meaning, of course, gets one to think deeper about where the origin for all the things we take for granted actually came from. The ingredients for an apple pie may include things like apples, flour, sugar, eggs, salt, etc. But where to these ingredients come from? To get these ingredients you must first invent a universe with laws like our own, that can lead to the formation of galaxies full of stars, which can fuse hydrogen atoms into heavier elements, which can then form planets. Some of these planets must have conditions that allow life to arise, which can evolve into things like apple trees and chickens (for the apples and eggs respectively) and for the evolution of intelligent beings that can put them together to make an apple pie. Some of the ingredients, like the salt (NaCl) and water (H2O), are relatively easy to produce, and just require the hundreds of millions of years necessary for stars to produce the heavy elements oxygen, sodium, and chloride (the hydrogen for the water was produced in the Big Bang itself). The other ingredients require billions of years in the making, for life forms and their evolution to take place. How brilliant and wonderful and simple a statement to make. Many people around the globe, that continue to honor and remember Carl make it a tradition to eat a slice of apple pie on Carl Sagan day. I know I plan to have a piece this year!

Sagan was an Astronomer, and one of the first astrobiologists. He was involved in important scientific research on the atmospheric composition of Venus and Mars. He played a major role in the Viking mission to Mars and the Voyager probes to the outer solar system. And, of course, he made communicating scientific findings to the public, and demonstrating the importance of the scientific process, a priority.

When I was growing up, the book Cosmos and the mini-series, by the same name, came out. Both were inspiring, thought provoking, and in some ways life altering, and Sagan tackled everything from Astronomy, evolution, the brain, and the importance of being skeptical of pseudoscientific claims. He had a mesmerizing way of delivering his message with intelligence and passion. The TV series was recently redone by Astrophysicist and science communicator, Neil Degrasse Tyson, who didn’t attempt to remake the original episodes, but who did an excellent job of continuing on where Sagan left off.

Sagan also wrote quite a few other excellent books. These included, “The Dragons of Eden”, “Broca’s Brain”, “The Demon Haunted World”, and “Billions and Billions”. These books really fueled my scientific curiosity growing up, as I’m sure they have done for many others who grew up to love science. After all these years, his books are still worth reading, if you haven’t done so already. He also wrote the science fiction novel, “Contact” that was made into a motion picture in 1997 with actress Jodie Foster. In the novel he attempted to show what first contact with an advanced alien species might be like.

I did have the opportunity to see Carl Sagan in person on one occasion. At the time I was a chemistry major in the mid-1980s at The University of Missouri – St. Louis. Sagan came to deliver a lecture to our university on the dangers of nuclear war and the importance of nuclear disarmament. He was a great dynamic speaker and the lecture hall was completely full. I think, at the time I was hoping he was going to talk about astronomy, but in retrospect I now understand the importance of his social concerns for our future and continued existence.

Sagan also introduced me to the concept of scientific skepticism, at a relatively early age. He was critical of how to tell the difference between science and pseudoscience (something now called the demarcation problem). He showed us that there are no beliefs that should be immune to skeptical inquiry, including religious belief. He came up with the “Baloney Detection Kit” that everyone should have in their skeptical toolbox.

Carl passed away right when the first exoplanets were just being discovered. Now we know of more than 1000 planets that circle other stars. We have made a much more thorough exploration of Mars and the moons of the outer solar system. We have strong evidence for liquid water deep under the crust of the moons Europa, Enceladus, and Ganymede. There is liquid methane on Saturn’s moon Titan. These discoveries make the possibility for life in our outer solar system a little more likely, and for life outside of our solar system very likely, by the sheer number of planets in our galaxy alone. At the same time the skeptical movement has gained momentum and is going strong. We have learned more about cognitive psychology and our innate biases and predisposition towards distortions of memory and perception. Flaws we must recognize in ourselves if we are to take the first steps to learn to become a more rational species and rise out of our superstitious past. I believe Carl would find all this fascinating and exciting. We could really use Carl’s wisdom now, but at the very least we still have him with us in the form of his writing and video.     

Happy Carl Sagan Day. Have some Apple Pie and be sure to learn something new today!
References and other items of interest:
1. Carl Sagan Wikipedia article: https://en.wikipedia.org/wiki/Carl_Sagan
2. Article detailing the “Baloney Detection Kit”: https://www.brainpickings.org/2014/01/03/baloney-detection-kit-carl-sagan/
3. Trailer for the movie “Contact” based on the book by Carl Sagan: https://www.youtube.com/watch?v=jl7Xe80_0MY
4. The Demarkation Problem on the Rationally Speaking Blog.
http://rationallyspeaking.blogspot.com/2013/08/philosophy-of-pseudoscience.html
Also check out the excellent “Rationally Speaking” podcast. The current host is Julie Galef, and excellent skeptic and teacher of all things rational! The previous host was Massimo Piglucci and scientist and philosopher and all around brilliant guy. Well worth checking out!
5. The Rationally Speaking Podcast with Julia Galef: http://rationallyspeakingpodcast.org
6. Some great Julia Galef youtube videos on rationality: https://www.youtube.com/user/measureofdoubt
7. Massimo Piglucci’s homepage: https://platofootnote.wordpress.com/massimo-central/
8. My contemplations on the possibility of what it would take for life to evolve on Titan:
http://darwinskidneys-science.com/2015/08/05/musings-on-the-biochemistry-on-saturns-moon-titan-part-i/

EMMA knows the secrets of your past – but will she tell?:

How molecular relics in your cells tell the story of our common origins.
By Rich Feldenberg

tRNA

In “Emma”, Jane Austin’s classic Novel, Emma Woodworth is described as handsome, clever, and rich. She takes to matchmaking, perhaps overestimating her abilities, and in doing so a variety of humorous and near disastrous calamities ensue. Of course, all ends well for Emma and her friends in the Novel. In this article we will examine a different sort of EMMA, but there may be some analogy to be found that even the brilliant Ms. Austin could not have foreseen. EMMAs is my acronym for Evolutionarily Modified Molecular Artifacts. I have used it in place of what has previously been referred to by some as molecular fossils. Fossil has the implication of something long dead, now extinct, and not seen in the world for many ages. Besides not being precisely what is meant by molecular fossil, when used by molecular biologists or astrobiologists, molecular fossil already has another meaning when referring to molecular or chemical remnants of past life. EMMAs may be a more appropriate term since it refers to molecular parts of still living systems that still display some resemblance to their more ancient and primitive forms. In this article we’ll explore a few examples of EMMAs and see what they can tell us about our distant past and the origin of life on earth. Austin’s Emma says “seldom, very seldom does complete truth belong to any human disclosure; seldom can it happen that something is not a little disguised or a little mistaken”. It is the nature of Evolutionarily Modified Molecular Artifacts, that their true nature is more than a little disguised and has traditionally been more than just a little mistaken. Lets look at the evidence that these living artifacts may give us a glimpse at a truth about our distant past, where we came from, and our common origins with our fellow living inhabitants on planet earth.

There are a number of critical biological molecules that are common to all life forms on earth today, and that have some unusual properties suggesting a common origin arising from more primitive precursor molecules. With this in mind, we’ll look at the common molecule ATP and the coenzymes NAD, and Acetyl Coenzyme-A, and finally the catalytic site of the protein synthesizing ribosome, which is perhaps the most fundamental molecular machine of any living cell. We’ll see that these examples also hint at a previous and now lost stage of life known as the RNA world, that preceded the Last Universal Common Ancestor (LUCA) of all living things on our planet today. To continue to stretch our Jane Austin analogy just a little further, we might imagine that the RNA world played matchmaker, in world long lost in deep time, and successfully paired DNA and protein, the two major biomolecules of life in our modern world. EMMAs demonstrate the remnants of that world before the matchmaking. Over evolutionary time they have been mesigned in their original forms, and re-mesigned into their current disguised forms. Like children who can not imagine a world before they were born, or before their parents existed, we too have a difficult time looking past the DNA/protein paradigm and into the RNA world.

Just like any good Austin Novel there are many interesting and complex characters. Some of the important players in our story of life on earth include molecules that contain pieces of ribonucleic acids (RNA). The first we’ll meet a key character known as adenine triphosphate (ATP). We will then be introduced to several of the coenzymes – small organic molecules that are necessary for the function of larger enzyme complexes. An finally we’ll become acquainted with one of the classic characters on life’s busy stage, the active site of the ribosome, which catalyzes one of the most fundamental reaction of the cell – the peptide bond to build protein. As stated above, each one of these molecules contains an RNA component, even though none of them are used to store or transfer genetic information. They are all involved in important biochemical reactions that have traditionally been thought to be performed only by protein enzymes. As we will see, the catalytic site of the ribosome relies on RNA exclusively to catalyze it’s fundamental reaction, and is therefore a ribozyme (RNA enzyme). These examples, and many others that we won’t describe today, appear to provide evidence of a long lost RNA world, with protein eventually evolving around the RNA core to assist and improve its biochemical efficiency.

First let’s look at the simple ATP molecule, which is well known to serve as the energy currency of the cell. It functions to power chemical reactions by transferring energy from its high energy phosphate bonds. It contains the base adenine, bound to the pentose sugar ribose. Ribose is the same sugar used in RNA (ribonucleic acid). The sugar ribose differs from the sugar deoxyribose (the sugar of DNA) only in the presence of a hydroxyl (OH) group at the 2-prime carbon. DNA does not contain this 2-prime hydroxyl group.

ATP is produced by the metabolic processes of glycolysis, the Kreb’s cycle, oxidative respiration, and by light powered photosynthesis, but is used in a multitude of reactions to provide the energy necessary to drive those reactions in the desired direction. Why should it be necessary that this energy storage molecule is a nucleotide? Could this be a hint that it’s important role began at a time when RNA played a much more central role in biology than it does today? Is the adenine now just a left over of the original mesign?

ATP
ATP – the energy currency of the cell.

Let me now introduce you to the charming NAD. Nicotinamide Adenine Dinucleotide(NAD) is a coenzyme that is composed of two ribose containing nucleotides linked together by a diphosphate connector. One of the nucleotides is adenine, just like that found in RNA, and the other nucleotide contains the non-RNA base nicotinamide. Being a dinucleotide, again should make us appreciate this coenzyme’s primitive origins.

NAD_drawing
Nicotinamide Adenine Dinucleotide (NAD)
The Adenine base is on the bottom half and the Nicotinamide is on the top half.

Nicotinamide is converted from nicotinic acid to its amide form. Nicotinic acid is also known as the vitamin niacin. The name was changed to niacin due the concern that people would confuse the nicotinic acid with nicotine and falsely believe that nicotine had nutritional health benefits. Nicotinic acid and nicotine are chemically distinct molecules, although they both share a pyridine ring structure- which is an aromatic heterocyclic ring with nitrogen at position 1 (see below). Both nicotinic acid and nicotine have their own distinct biological effects. Of course, nicotine is produced by the tobacco plant, but is not produced by animal cells. Nicotinic acid is found in all living cells, whether they are animal, plant, or single celled bacteria.

AT-GC_base_pairs

Purine_bases

pyrimidine_bases


Nicotinamidenicotinic_acid_structureNicotine-2D-skeletal
Chemical similarities between Nicotinamide (part of NAD) on the left, Nicotinic acid (Niacin) in the middle, and Nicotine (harmful carcinogen) on the right.

Nicotinamide Adenine Dinucleotide (NAD) is necessary for the operations of a wide variety of enzymes in all cells. The NAD molecule can be in either an oxidized form (NAD+) or a reduced form (NADH), and is therefore an important component of many oxidation-reduction reactions in the cell. It can transport electrons in its NADH form, or take them away in its NAD+ form. Since cell metabolism is, in large part, the process of extracting energy from biomolecules like sugars and fatty acids – in other words oxidizing these molecules in a slow and controlled way – NAD is important for the function of many enzymes found along these these metabolic pathways in the cytosol and mitochondria in eukaryotic cells. In the mitochondria NADH becomes oxidized, as electrons flow down the electron transport chain. The resulting H+ (proton) is pumped across the cellular membrane, creating a proton electrochemical gradient, which then is used to produce ATP – to be used to power other non-spontaneously occurring chemical reactions.

NAD_ball_and_stick_model
Ball and Stick chemical model of NAD

The coenzyme known as Acetyl-CoenzymeA , like NAD, also contains the nucleotide adenine. Connected to it is a molecule with a thiol group (SH) at its end. This molecule participates in important chemical reactions that require the transfer of an acetyl group (a methyl bonded to a carbonyl – see below).

acetyl group

         acetyl group

Many steps in key chemical pathways involve acetyl transfers to build or break down molecules. The sulfur group in Coenzyme A can chemically attack the acetyl group of another molecule, remove it from that molecule, and thereby take it for use in a multitude of biochemical reactions. In the process Coenzyme A becomes Acetyl Coenzyme A, and can be recycled back to Coenzyme A once it released the acetyl group at the right time and place. It is an important part of enzymes involved in glycolysis and the Krebs cycle – both chains of reactions that break down glucose to create ATP. Gene expression can also be regulated by acetylation of histone protein, telling the cell which genes to transcribe and which need to remain silent in a given cell type. It is also used to create the neurotransmitter acetyl-choline from choline.

coenzymeA

Conenzyme A. To become Acetyl-Coenzyme A, an acetyl functional group is attached to the thiol group at the far left end of the molecule. In this way, Acetyl-Coenzyme A can transport a carbon atom to be used in other chemical reactions.

Our true hero is the ribosome, the site of protein synthesis, and common to all modern cell types, although, the molecular structure differs enough between prokaryotes (single celled organisms like bacteria and archaea) and eukaryotes (more sophisticated cell types like that seen in animal or plant cells) that these differences can be exploited by certain antibiotics which target prokaryotic ribosomes, but leave the eukaryotic ribosomes unharmed. Even the mitochondria found in animal and plant cells have their own ribosomes that resemble prokaryotic ribosomes more than eukaryotic ribosome found in the cytoplasm of those same cells. The production of protein is perhaps the most primitive and basic metabolic function of all living cells. It came as a huge surprise to scientists when they learned that the active site of the ribosome (where the peptide bonding reaction takes place – the peptidyl transferase reaction) is composed only of RNA and no protein at all. Additional structural studies have confirmed that it is the RNA that catalyzes this basic cell reaction.

protein_synthesis

Ribosome1

This would seem to support the notion that RNA played the major role in the biochemistry of the most primitive life forms. Ribosomes today are complex molecules, made of multiple components, some of which are ribosomal RNA and other parts are protein – it is therefore a ribonucleoprotein. The protein portions seem to assist the ribosome in doing its job more efficiently.

The examples given above reveal the important role that RNA molecules play in cellular biochemistry. The fact that some of the basic process of life rely on these RNA containing molecules lends support for the RNA world hypothesis. Except for the ribosome where the actual catalytic site is still a ribozyme, the other examples don’t use the RNA portion for the vital catalytic role, but may possibly have done so in the distant past. The presence of the RNA still retained in the coenzyme may offer proof that it is a molecular fossil – or as I prefer an Evolutionarily Modified Molecular Artifact (EMMA). To paraphrase Jane Austin, when referring to the RNA that lays hidden at the core of many of our most rudimentary metabolic processes, which may have served a grander role in a far distant past, and which now has relinquished it’s primary role for one of a more modest, behind the scenes assistant, “The sweetest and best of all molecules, faultless in spite of all her faults”. In future articles we can examine some other examples of EMMAs, such as additional types or ribozymes and riboswitches.

References:

1. Article on Mesign in Nature (also linked to within this article):
http://darwinskidneys-science.com/2015/07/08/another-clever-mesign-brought-to-you-by-mother-nature/

2. Nicotinamide Adenine Dinucleotide (NAD) Wiki article.
https://en.m.wikipedia.org/wiki/Nicotinamide_adenine_dinucleotide

3. Acetyl Coenzyme A Wikipedia article.
https://en.m.wikipedia.org/wiki/Acetyl-CoA

4. “The RNA World” Gesteland, Cech, and Atkins. Second Edition, Cold Spring Harbor Laboratory Press. 1999.

5. “Molecular Biology of the Gene” Watson, Hopkins, Roberts, Steitz, Weiner. Fourth Edition, 1987.

6. Life as we don’t know it.   “Musings on the Biochemistry of Saturn’s Moon Titan”.

Part II: Musings on the Biochemistry of Saturn’s Moon Titan

Part II: How can we envision solutions to the challenges for life on Titan?

By Rich Feldenberg
Titan_and_rings

Definitions:
Non-Polar Liquid Solution – NPLS
Polar Non-Aqueous Liquid Solution – PNAL
Titanoids – terpenoid-like molecules used by life on Titan. They provide the subunits (monomers) necessary to make large molecules (polymers), the way amino acids make proteins on earth.
Protiteins – These molecules are build up from the smaller titanoid subunits, and serve the role of proteins for Titan life forms.
Genoids – steroid-like molecules that have been naturally selected by life on Titan to store the genetic information, similar to nucleotides on earth. They can “base pair”, based on complementary physical fit with another specific form of genoid.
Titanite – life forms found on Titan.
Reverse lipid bilayer – a molecular membrane on Titan in the PNAL scheme that has its hydrophobic tail facing the methane solution outside the cell, and its polar head facing the cell interior to remain in contact with the polar solvent concentrated in the cell.

In Part I of this article we discussed the special properties of water that makes it so important for life on earth, and the properties of liquid methane on Titan that make it a poor substitute for life as we know it. So while in Part I we focused on what the problems are for Titan biochemistry to exist, Part II will look at some theoretical solutions to those problems. For methane to be the fluid of life, would require life to take on an entirely different character from what we know. Methane’s non-polar physical character presents major hurdles that may prevent interesting biochemistry from taking place in a such a non-polar liquid solvent (NPLS). While liquid methane is abundant on the surface of Titan, and undergoes a methane cycle, similar to the water cycle on Earth, a non-polar solvent for life would seem problematic at best, and impossible at worst. For that reason we will also consider some other options for life on Titan. We’ll look at the possibility of a Polar Non-Aqueous Liquid Solution (PNAL), that might provide a way for Titanites to survive and evolve by creating an internal environment friendly to polar chemical reactions, like those seen in Earthly life forms. As I said in part I of the article, “We will stick, as much as possible, to the rules of organic chemistry, but Warning, wild speculation ahead”. Ready to jump into a lake of liquid methane? Here we go!

The Non-Polar Liquid Solution (NPLS):
Lets start with the Non-Polar liquid solution (NPLS) possibilities. If we accept methane as our solvent because it is the liquid that happens to be flowing on Titan’s surface, then we’re faced with a difficult dilemma. Methane is a non-polar compound. It’s dipole moment is zero, zip, nadda, in other words, it has no permanent charge separation within the molecule itself – unlike water which has a relatively large dipole moment of 1.85 D. So just like the adage, water and oil don’t mix, on Titan we’re dealing with oil so the things that will dissolve in methane are also going to be non-polar substances or hydrophobic. The kinds of biomolecules that make life possible on earth would not do well in that “hateful” environment. In fact, the very thing that makes life interesting on Earth is that the molecules of life are very reactive, have groups of atoms in the molecules themselves, that are polar and therefore reactive with other polar molecules. This provides the basis of most of the biochemistry of Earth life. So on Titan, in the NPLS scenario we would not expect to find things like Earth-like DNA or proteins. So how could it work?

alkane

We would need non-polar molecules so that they could remain in a methane solution. Due to the extreme cold on Titan, any polar molecules would have too stable of an interaction with each other and precipitate out of solution as a solid. In other words, DNA, and other similar molecules would crystalize out. Not really a very good way for life to get started. On Earth we do have lots of non-polar biomolecules in the form of waxes, oils, and fats. The biochemistry on Titan, in the NPLS would have to be made of something like that. Life also seems to need small building block materials (monomers) that can be polymerized together to make large biomolecules. We need to find non-polar compounds that can, in principle function that way. Two possibilities could be substances similar to terpenoids or steroids.

Let’s look at terpenoids first. Terpenoids are a class of lipids that are made out of multiples of 5 carbon units. Their precursor molecule is isopentenyl diphosphate – a 5 carbon compound attached to a diphosphate group. By using this as the basic building block, you can build up a larger sized molecule with the isopentenyl as the basic unit. Since Titan’s oceans are methane with some ethane, we might imagine our basic building block to be a 2 carbon unit – made from the ethane. That way our biomolecules would be large with an even number of carbons because they are built up from multiples of two. Instead of terpenoids, we’ll call these molecules titanoids, and they could perhaps function in the role of amino acids, being used to build larger biomolecules, the equivalent of proteins – we’ll call these protiteins (pronounced Pro-Tie-Teens). There could be different titanoids used – like the 20 different amino acids on Earth – each with a different non-polar side chain.

Next lets look at the steroids. These are a diverse class of molecules which all have a basic 4 part ring structure. All steroids are themselves derived from a terpenoid compound called lanosterol. The rings, which are labeled A, B, C, and D, can themselves be altered in many ways to give the wide variety of steroids found in plants and animals. Having a hydrocarbon ring system, like in the steroids found on Earth, produces an essentially non-polar and hydrophobic compound. Like terpenoids, they could mix easily in the liquid methane environment found in the cells of Titan organisms. The rings themselves can be boat-shaped if saturated, but flat if aromatic (meaning they have double bonds like benzene that provide an aromatic resonance).

We might imagine the non-polar and very cold environment on Titan to favor a kind of stacking of these rings. They might be produced to fit neatly on top of one in other, so that instead of have a linear molecule composed of many monomers, like Earth protein or DNA, on Titan we might have layers or thick sheets of molecules that are stacked on top of each other, held in place by their Van der Waals forces, to provide the structure. The weak Van der Waals forces would keep the molecules, essentially glued together. This would provide a very different and alien kind of molecular structure than earth organisms which put molecules together with covalent bonding.

We also need some type of information storage molecule to allow a Darwinian type of evolution to take place. We might also imagine steroid-like molecules serving the function of information storage that DNA and RNA can serve on earth. There would not be hydrogen bonding between nucleotide bases possible in the cells hydrophobic methane protoplasm. Instead several different steroid types (lets call these genoids to indicate they form the genetic material based on a steroid structure) might have been chosen in which one molecule seems to make a very good physical fit with one of the other chosen genoid molecules – essentially a lock and key type of interaction based on complementary shape. No hydrogen bonding would be necessary, just the close fitting together of genoid 1 with genoid 2, and genoid 3 with genoid 4. That way there is a template for copying the genetic material, and way to store information in the genoid 1, 2, 3, and 4 code. The back side of the genoids might contain functional groups, such a methyl groups, that would create a steric hindrance so that they can only fit together in the desired base-pairing way.

There could be only 2 genoids, although that would not allow for much coding information, or there could be more than 4. So for example, a “codon” with the genoid sequence 111, might code for titanoid A, and the codon with genoid sequence 112, might code for titanoid B, and so on. The genoids might stack on top of one another but fit together with their complementary sheet, which forms the template for replication. Because dispersion forces, which are terribly weak, are holding the works together, this would only be possible at the extremely low temperatures, like those found on Titan. If the temperature was raised the molecules would fall apart and the organism would essentially melt away. Even the temperature of dry ice (solid carbon dioxide) with a temperature of -109F, would be hot as hell for our friendly little Titanites.

genoid A&B

genoid base pair

Somehow we would have to have a mechanism that allowed the genoid code to be translated into the titanoids to make some sort of polymer structure. Since hydrophobic chemistry is rather boring – I mean only in the sense of being unvaried, not that it is stuffy in any way – because there is no nucleophilic substitutions or eliminations, or so forth, molecular reactions other than simple dipole-dipole and dispersion interactions, would probably have to take place through radical reactions, using some energy source to produce the free radicals.

methane

Polar-Non Aqueous Solution (PNAL):
The PNAL scenario would work by allowing the cells on Titan to essentially evolve to become containers of weakly polar solvent, which keep the polar solvent inside the cell membrane, and the non-polar methane on the outside. There would have to be a way to pump in the polar solvent from the environment and concentrate it in the cell, preventing it from leaking back out. The advantage here is that we then can allow the much more interesting types of chemistry to occur in the cell, with reactions like the nucleophilic substitutions, additions, and eliminations, which can be used to build interesting molecules and metabolize others. This type of life might seem slightly more familiar than the NPLS, that we considered above, but would still be a very alien “life as we don’t know it” variety. We might have to imagine the cell membrane to be a kind of reverse lipid layer, instead of the lipid bilayer found in Earthly cell membranes. The lipid bilayer of Earthly cells has the lipid tails in contact with each other, sandwiched in, while the hydrophilic phosphate heads are facing outward on each side, making contact with the aqueous environment of the interior cell on one side and the water external to the cell on the other side. The reverse lipid layer would have the hydrophobic tail sticking on the outside to interact with the methane environment, but the polar head region (like a phosphate group) on the inside to interact with the polar solvent concentrated in the cell.

The solvent here would still need to be an almost non-polar substance with a very low dipole moment. Any truly polar substance – one having a high dipole moment – would have too much molecular interactions with other molecules of its kind, and would solidify at the temperatures found on Titan’s lakes and rivers. Water has a dipole moment of 1.85D, which is way too high for this environment. In fact, water on Titan’s surface forms rock hard geological structures. One substance that might work would be Arsine (AsH3) which has a dipole moment of 0.20. Arsine, with one Arsenic atom and three hydrogen atoms, might conceivably be a substance that could be found or produced on Titan since Arsenic (As) and Hydrogen (H) are both common elements. The freezing point of Arsine is -168.16F, which is pretty cold but Titan is -291.1F, so this would only work if there were some warmer spots on Titan where there is geothermal energy reaching the surface. If true though, Titanites would drink arsenic the way we drink water! The reverse would be equally true, water would be a deadly poison to their delicate machinery of life.

Arsine-underside-3D-vdW

Arsine

Perhaps a better choice of polar solvent would be Perchloryl fluoride, with a dipole moment of only 0.023D and a freezing point of -234F. This is still a little short of the -291.1F of Titans Lakes, but fairly close so perhaps there could be some “hot spots” where the temperature is a balmy -234F, and the perchloryl fluoride would flow free! Perchloryl fluoride is a molecule made of one chloride, one fluoride, and three oxygen atoms, so it may be something that life on Titan could find or produce and concentrate in their cells. The low dipole moment might still mean that other polar molecules would not react well with it, and there would be the risk of other polar biomolecules freezing out of solution. Getting caught in a slightly cooler zone for PNALs would spell disaster for these little Titan cells, as they would crystalize, whereas the NPLS would seem well suited for the extreme cold of a world far from it’s star.

Since the temperatures are so cold we might also need to consider molecules that are extremely reactive – even too reactive for Earth life. We might consider acid halides in place of the usual functional groups like carboxylic acids, alcohols, esters, and so on. On earth acid halides are too reactive – the halide (such as chloride) would be too good of a leaving group – and they would therefore have no stability. On Titan, however, where the temperatures are so cold the acid halides, like acid chloride for example, might be longer lived so that they might have more stability and could be controlled by cellular process to take part in chemical reactions at the appropriate times.

acyl_chloride

Whether any of this could really play out on Titan or a similar world, I don’t know. This is the fun of pure arm-chair astrobiology. Perhaps some of these ideas could be tested to some degree in the lab. Our only real way of knowing if Titan could support some form of life is to send robotic probes that can go there and conduct the right experiments. I sure hope that a mission like that will happen sometime in our lifetimes. Certainly finding life as we don’t know it would increase our understanding about what life is, and would ultimately mean that life must be very common in the universe if it can manage to find ways to exist on worlds as different as Earth and Titan.

References:

1. Titan: Wikipedia; https://en.m.wikipedia.org/wiki/Titan_(moon)

2. Freezing and Melting Points of some common liquids:
http://www.engineeringtoolbox.com/freezing-points-liquids-d_1261.html

3. “Selected values for electric dipole moments for molecules in the gas phase”, United States Department of Commerce; http://www.nist.gov/data/nsrds/NSRDS-NBS-10.pdf

4. Organic Chemistry; John E. McMurry, 8th edition. ISBN-13: 978-0840054449
5. See my article on Silicon Based Life forms (another variety of life as we don’t know it):
In this case it would presumably be water dependent but not based on carbon.
“Why the Horta would not have looked like a rock monster”;
http://darwinskidneys-science.com/2015/06/18/why-the-horta-would-not-have-looked-like-a-rock-monster/

6. See Part I of this article. “Musings on the Biochemistry of Saturn’s moon Titan, Part I”.
http://darwinskidneys-science.com/2015/08/05/musings-on-the-biochemistry-on-saturns-moon-titan-part-i/

7. McLendon, Christopher, et al. “Solubility of Polyethers in Hydrocarbons at Low Temperatures. A model for Potential Genetic Backbones on Warm Titans”. Astrobiology, Volume 15, Number 3, 2015.

Musings on the biochemistry on Saturn’s moon Titan. Part I

Part I: what makes potential life on Titan such a challenge?
By Rich Feldenberg

Titan_and_rings

As we have been repeatedly surprised by the discoveries made by our robotic explorers over the last few decades, it is certainly clear that our solar system is much more interesting and diverse than we ever previously imagined. There appears to be at least several objects among our cosmic neighbors that show promise as potential habits for life outside of Earth. Some of the prime real estate in this respect would be Mars, Jupiter’s moon Europa, and Saturn’s moon Enceladus. Both Europa and Enceladus show evidence of having liquid water, vast oceans in fact, beneath their frozen crust, and Mars most likely had liquid water on its surface billions of years ago, and perhaps still has liquid water somewhere below the surface. Even Venus may have been covered in water in it’s early history only to evaporate it’s oceans away due to an out of control green house effect. In addition to these, there are other candidates moons and even dwarf planets, that may be home to liquid water beneath a frozen surface, and therefore also a potential abode for living things.

Everything we know about life on earth points to the importance of water in the origin and maintenance of life. If we find life on any of these worlds it will probably be “life as we know it”, in the sense of having a similar biochemistry to earth life with a dependance on liquid water. In fact, it would probably be a consequence of being water based that would contribute to the close similarities in molecules like DNA and proteins, that would most likely be present on a watery world. There may certainly be some important and interesting differences, such as using a different genetic code to specify amino acids, perhaps having a different chirality (i.e. Right handed amino acids instead of left handed ones), and possibly having a different set and number of amino acids, but the basics of DNA to code and store information and proteins made of amino acids to carry out metabolism, will likely look like a odd version of what we have here on earth. But, there may be one place in our solar system where the potential for “life as we don’t know it”, also has the potential to exist. That place is Saturn’s moon Titan.

Titan is unique in the solar system in a number of important respects. For a moon its quite big, being the second largest moon in the solar system – only Jupiter’s moon Ganymede is larger. Titan has twice the diameter of earth’s moon. If it was in orbit around the sun instead of Saturn, it might be considered a planet unto itself. It is also the only moon with a thick atmosphere, as dense as the atmosphere down here on earth. Like Earth, the atmosphere on Titan is primarily nitrogen, but unlike earth there is no oxygen to breath. But perhaps the most remarkable thing about Titan is that there is liquid on it’s surface. Besides Titan, only the earth is known to have a large volume of surface liquid today. As we said before, Mars probably had liquid water on it’s surface billions of years ago that has since evaporated away. Of course, the earth has vast oceans of liquid water, but the liquid on Titan is not water, it’s liquid methane. There are lakes of methane, and evidence for rain and rivers of the stuff! We think that liquid is necessary for the chemistry of life to take place. In solids the molecules are too fixed to react much and gases are usually to dispersed for reactions to have a high chance of occurring, and are difficult to contain. A major difference between Earth and Titan, however, is that Titan is cold – really cold. The surface temperature on Titan is -179.2 degrees celsius or -291 degrees fahrenheit. Now that’s pretty darn cold, and Titan would be even colder if not for the greenhouse effect of methane in its atmosphere. It’s both the extreme cold and the fact that methane is the liquid we have to deal with, that makes any type of biochemistry onTitan so challenging to consider.

Titan_Surface

Water has properties that make it an ideal solvent to dissolve the molecules of life into solution. Could liquid methane serve a similar role on the frozen moon Titan? What would it take for life to find a way to use methane as a solvent for the molecules of “life as we don’t know it”? Let’s look at some of the possibilities for a methane dependent life form that lives in the conditions present on Titan. We will stick, as much as possible, to the rules of organic chemistry, but Warning, wild speculation ahead.

Let’s first look at the major differences between water and methane as your choice of solvent. Every school child knows that water is H2O – made up of two hydrogen atoms covalently bonded to an oxygen atom. It is not a linear molecule, however, but has a bent shape. This creates an electric dipole moment in the molecule, meaning that one side of the molecule is partially negatively charged (the oxygen in this case), and the other end, facing the hydrogens, has a partial positive charge. The electric dipole moment can be measured and it’s value is placed in units of the debye (D), with water having an electric dipole moment of 1.85D. It is this dipole, combined with the fact that oxygen and hydrogen are extremely common throughout the cosmos, that makes water such and interesting, useful, and seemingly indispensable substance. Most organic molecules are electrically neutral – they have no formal charge – but many of the functional groups in the organic molecules do have an uneven distribution of electron sharing, creating a partial charge separation in the chemical bonds between certain atoms. For example, in the carboxyl group there is a partial negative charge on the oxygen and a partial positive charge on the carbon. This means that the carbon can act like an electrophile (attracted to electron rich atoms) and reacts with a partially negative charged molecule, and a nucleophile (attracted to an electron poor atom) will tend to react with the carbon. The reactions due to these kinds of attractive forces are called polar interactions, and it is these types of interactions that create interesting chemistry, and life is all about interesting chemistry.

hydrogenbonding_water

Water can form hydrogen bonds with other water molecules, as in the figure above. Because of waters dipole properties it will interact strongly with other molecules that contain polar covalent bonds, and this allows them to be soluble in a watery solution. This is energetically favorable for polar molecules, because even though they cause the hydrogen bonds of the water matrix to break as they take up space in the solution (energetically unfavorable), they then form polar bonds with the water to replace the broken bonds (energetically stable)- so in a sense, no harm done. This allows the polar molecules to be soluble in water solution, and this is what you want for your biomolecules if you’re a life form – soluble molecules, not ones that precipitate out of solution. Water helps to stabilize polar molecules and is very important for maintaining the proper shape and structure of important biomolecules like DNA and proteins.

Non-polar molecules, on the other hand, really don’t do well in water and they will not go into solution. The non-polar molecules also break the waters hydrogen bonds since they have to fit in the water matrix, but since they are non-polar they can not replace those bonds, and so remain energetically unfavorable. They will tend to form a separate layer from water and stick together to minimize their interaction with water molecules. For this reason, the non-polar molecules are called hydrophobic, which means water fearing or water hating.

Water is also great at dissolving important salts in solution, like sodium chloride (NaCl) for example. The polar nature of water will break the ionic bonds between the sodium atom and the chloride atom apart, and the sodium will be surrounded by a sphere of water with it’s partially negative oxygen atoms facing it, while the chloride will be surrounded by a sphere of water with it’s partially positive hydrogen atoms facing it. Inorganic ions from salts like sodium chloride, potassium chloride, calcium phosphate, magnesium sulfate, and others are vital to the workings of enzymes and stabilizing biomolecules. Would these ions be able to exist in a non-polar methane sea?

Unlike most organic molecules, one group that is non-polar and water hating are the hydrocarbons. Methane is the simplest hydrocarbon, having just one carbon atom covalently bonded to four hydrogens. There is no dipole moment in the methane molecule. Carbon and hydrogen both have approximately the same amount of pull for the electrons. Even if that weren’t so, the symmetrical tetrahedral shape of methane would still cause the polar bonds to cancel each other out. Hydrocarbons with long chains of carbons are also non-polar. Propane is a hydrocarbon with 3 carbons and 8 hydrogens, and octane is one with 8 carbons and 18 hydrogens. Methane is the major liquid on Titan, with some ethane (2 carbons) thrown in for good measure. Hydrocarbons don’t undergo polar reactions. That’s one reason that they can remain liquid at such low temperatures where other organics would freeze solid – they interact so weakly that it is difficult to get them to interact enough that they become solid. These non-polar molecules do have their own very weak interactions, however. They are subject to non-polar forces called Van der Waals forces. These results from a weak induced dipole, as one molecule gets close to another. Say you have a mixture of methane molecules. There is no permanent dipole, but when the hydrogen of one methane gets too close to the hydrogen of another methane the electron of the first will repel the electron of the second one, and that creates a tiny fleeting attraction between the two molecules. It becomes more pronounced at very low temperatures where the heat energy of motion is not too great. At temperatures that we find comfortable, these minuscule interactions are easily overwhelmed by all the thermal motion. If you have very long hydrocarbons, or other non-polar molecules, then the combined Van der Waals forces acting along the different parts of the molecule can make the effect more significant. This would possibly be a very important force in any Titan life form. On Earth, Van der Waals forces certainly have their important places, and can be important in many DNA-protein interactions and some protein-protein interactions, but many of the interactions between biomolecules rely on polar chemistry to create covalent bonds, and this might be difficult to achieve for a living thing on Titan.

In part II of this article we will examine two possible solutions for Titan biochemistry – What I’m calling The Non-Polar liquid solution (NPLS) versus the Polar-Non-Aqueous liquid (PNAL) solution, or the NPLS vs PNAL. By solution, the meaning can equally double as referring to either a mixture of a liquid with another substance or an answer to a problem – either works fine.

References:
1. Titan: Wikipedia; https://en.m.wikipedia.org/wiki/Titan_(moon)

2. Freezing and Melting Points of some common liquids:
http://www.engineeringtoolbox.com/freezing-points-liquids-d_1261.html

3. “Selected values for electric dipole moments for molecules in the gas phase”, United States Department of Commerce; http://www.nist.gov/data/nsrds/NSRDS-NBS-10.pdf

4. Organic Chemistry; John E. McMurry, 8th edition. ISBN-13: 978-0840054449

5. Also see my article on Silicon Based Life forms (another variety of life as we don’t know it):
In this case it would presumably be water dependent but not based on carbon.
“Why the Horta would not have looked like a rock monster”;
http://darwinskidneys-science.com/2015/06/18/why-the-horta-would-not-have-looked-like-a-rock-monster/