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Secrets in the Salt: Expert Q&A

On July 31, 2009, Jack Griffith answered selected questions about the cellulose, DNA, and other organic bits found in 250 million-year-old salt deposits as well as the electron microscope technology used to discover these remains.



Q: What steps do you take to avoid contamination from cotton, paper, and other "modern" cellulose fibers in your samples and preparative techniques? Fay Goldblatt, Wilmette, Illinois

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Jack Griffith: Dear Fay,
In essence, our current method is to soak the crystals in a solution of chromic acid. On the program, you saw Smaranda Willcox doing this. This solution is so caustic that it will degrade any biological material into single atoms in minutes. If one puts a fragment of a leaf into this acid, it is destroyed completely. By soaking the crystals this way, any biological material stuck to the surface will be eaten away, leaving only the pristine salt crystal. Also, by submerging the crystal in the acid solution, we can assess its integrity. If there should be any cracks in it, the yellow acid will make these visible and alert us to not include it for further analysis. This way we assure that any inclusion we analyze has not been altered since its formation. This material is then treated in the most sterile manner and dissolved in ultra-ultra pure water and then processed for EM [electron microscope]. We do side-by-side controls of just the water and buffers used. We have never seen any such cellulose fibers as contamination in our routine experiments, which have been ongoing over 30+ years.

For more on this, see our 2008 paper in Astrobiology:

Jack D. Griffith, Smaranda Willcox, Dennis W. Powers, Roger Nelson, and Bonnie K. Baxter. Discovery of Abundant Cellulose Microfibers Encased in 250 Ma Permian Halite: A Macromolecular Target in the Search for Life on Other Planets.

Astrobiology. April 2008, 8(2): 215-228.

Q: Hello,
Is anything in the little air pocket time capsules "alive" (such as the bacteria)? Is the DNA able to be "read" in hopes of understanding the life-form it's from? Thank you! Anonymous

Griffith: Dear Anonymous,
The liquid inclusions trapped in the salt have been thought by some to contain dormant bacteria that can be made to come back alive when placed in modern bacterial media. We (Griffith lab) are not sure about this, since in nearly 100 independent visualizations of material present in these liquid drops, we have never seen a single bacterial cell, even ones that are disrupted. This agrees with current biochemical experiments that argue that protein molecules may degrade spontaneously over a time frame that would eliminate things like viruses and bacteria long before 250 million years had gone by. More work will be required to solve this question.

There are, however, some small scraps of DNA present in the liquid and in the solid salt. It appears that the salt environment is able to protect DNA from breakage. One of the most exciting projects in our laboratory at the moment is to work out ways of interrogating this ancient DNA—that is, reading its sequence. This would provide clues into its origin and, in a more grand way, the general nature of DNA a quarter of a billion years ago.

The bubbles in the fluid within the tiny cavities can be formed in differing ways. One is by shrinkage of the fluid if it was much warmer when "captured." Another is to have a gas dissolved in the brine when it was trapped at one temperature, and then the gas exsolves or comes out of solution to form a little gas bubble when the crystal and trapped fluid is cooler.

Q: There are numerous salt domes worldwide. When and what brought about their formation? Norm Green, North East, Pennsylvania

Griffith: Salt domes are caused when salt is buried and then rises up through overlying rocks, because the salt is less dense than the overlying rocks. It's trying to float, in a sense. When salt is buried more deeply, the greater temperature and pressure also make that salt behave more like a plastic material, at least over geological time periods. It can deform and change shape more easily, and this also makes it easier for the salt to move upwards (because of low density) through weaker points or areas in overlying rock.

As Mr. Green mentions, salt domes are found in several different parts of the world. The North American Gulf Coast is a well-known area of salt domes. There, as a salt layer was buried several tens of thousands of feet, the combination of low density, high temperature, and high pressure resulted in the more plastic salt rising through soft, overlying sediments. Like a very slow-moving bullet fired upward into thin layers of some material, the salt pierced the overlying layers and even bent the edges upward. Where these upward-bent edges of oil-bearing rocks abut the salt, an excellent reservoir can be found. Much of the early oil exploration around these features used high-precision gravimeters that could measure the gravitational effects of having a dome of low-density salt surrounded by higher-density sediments or rocks.

The Salado Formation in southeastern New Mexico is layered, with layers having thicknesses of a few feet. They are nearly flat-lying and are not buried deeply. The Salado is mildly deformed, but it has never reached the conditions necessary for forming salt domes. This is also part of the information that indicates these rocks remained unbreached by nature for 250 million years.

Note: This question was answered by Dr. Dennis Powers, University of Mississippi

Q: Would you have had access to the formation if WIPP [the U.S. Department of Energy's Waste Isolation Pilot Plant] had not already created access? How do you feel about TRU [transuranic] waste being stored in this formation? It seems relevant and odd to not mention the current use of the salt in this area. Thanks! Jamie, Brooklyn, New York

Griffith: Dear Jamie,
For nearly 80 years, the Salado Formation near the WIPP has been mined to produce potassium fertilizers. Active mines still exist that expose various parts of the salt beds. Nevertheless, these are production mines, and it is more difficult to accommodate scientific work. The WIPP is an operating disposal site, but it also has a mission to encourage scientific study where feasible. Hence the WIPP provides the perfect means of getting samples that have not been exposed to surface contamination for 250 million years.

My feeling about the site as a storage facility is that the very careful geologic studies by Dr. Powers, Dr. Roger Nelson, and others have shown that the formation this deep underground is not only very stable but extremely unlikely to be disturbed for the next tens of millions of years, far longer than the time required for this radioactive material to decay. Thus, given the options we have, it seems an ideal site. And the excavated salt can't be sold—competes with private industry.

Q: My daughter has just graduated from UNC-Chapel Hill. We love the place. My question: Why do we care about 250 million-year-old cellulose? What is it telling us? The company I work for is managing the WIPP. Take care. Richard Royer, Charlotte, North Carolina

Griffith: Dear Richard,
We, too, love Chapel Hill—a very cute village in my view. The finding of "native" biological molecules this old is important. By native, I mean that this ancient, 250 million-year-old cellulose behaves in a biochemical and structural manner just like modern cellulose: It looks like modern cellulose and is chewed up by enzymes that chew up modern cellulose. Hence, we can say that by discovering this material in the salt deposits, we have direct evidence of life that long ago. This makes it much, much harder for others to argue, for example, that the Earth is only a few thousand years old. Of course, we have fossils that date much farther back, but these are rock casts of plants, fish, or animals in which the biological material has long since been replaced by minerals.

In another line of argument, finding such ancient biological molecules opens the door for our further experiments to read the sequence information in the scraps of DNA that we have found and thus be able to interrogate very ancient DNA as to what it came from and perhaps learn about species that no longer exist on the planet. Smaranda Willcox and I are planning visits to sites in Europe where we may be able to collect salt crystals that date from intermediate times, say 5 to 20 million years ago. Such material could contain a great deal more biological material than what has been present in our very old salt. Thus by examining salt from different locations and times, we may be able to generate a time line of how biological material is preserved in the salt.

From yet another perspective, the finding of cellulose trapped for so long in a salt deposit on Earth, which attests not only to its resilient nature and its abundance whenever life is present, but also to the exceptional preservation qualities of salt deposits, can be instrumental to studying the presence of life on other planets. Our discovery came almost at the same time as the discovery of salt deposits on Mars. We were ecstatic at the implications. We had found the paper trail to life on Mars! If anyone at NASA reads this, we are very interested in looking at martian salt crystals, whenever they can bring some down to Earth!

Q: Hi,
In the salt program, you said that the cellulose you found in the salt crystals is the oldest direct evidence of life on the planet. But I've read about organic compounds called "biomarkers" that are billions of years old. What's the difference? Does the difference lie in your use of the word "direct"?

You also said you could do biological tests on the cellulose, such as you can't do with fossils, say. I'm just wondering: Have you done any such tests on the ancient cellulose yet? And if so, what have you found?

Thank you. Anonymous

Griffith: Dear Anonymous,
Indeed, there are older organic compounds on the planet—for example, oil. However, the molecules that comprise oil have been battered, heated, and compressed by geologic events to the extent that they no longer resemble what they came from. In our case, the cellulose molecules are intact and have not been altered to any significant degree from the time they were synthesized by some living organism—plant, bacterium, or alga.

We have done a series of tests. These are described in our paper in the journal Astrobiology (see full reference in first answer above). We hope to have a copy for readers to download. The most important test used the enzyme cellulase that degrades cellulose. Incubation of our ancient cellulose with this enzyme destroyed it in a few minutes.

In other studies, investigators have used amplification methods that replicate very tiny amounts of DNA found in even older salt. The difference is that the original DNA was not directly observed; they could examine copies made in the laboratory. That's very useful as well, but it's not a direct observation of the original material. Likewise, bacteria thought to be the same age as the cellulose were grown from cultures—in the laboratory. Again, the bacterial culture obtained was new and was supposedly a result of the ancient bacteria multiplying themselves. In our study, the original material from the crystals was directly visualized, and we directly observed what it contained: large amounts of cellulose and smaller amounts of DNA.

Q: I was fascinated to watch Jack Griffith's "Secrets in the Salt" segment on NOVA science NOW on July 28, 2009.

How did cellulose get into the saltwater solution that eventually formed the huge salt deposit from which you extracted halite crystals for examination?

I thought cellulose was a by-product of breaking down wood fibers through chemical and heat-based processes.

Does the 250 million-year-old DNA you discovered in the pockets containing saline solution from your samples actually come from ancient trees?

Thank you for taking the time to answer.

Yours sincerely, Kurtis Kitagawa, Ph.D. (in History, which explains why I am not up to speed on microbiology!), Ottawa, Ontario, Canada

Griffith: Dear Kurtis,
Our work is sort of very ancient history—at the electron microscope level. There are a number of ways that cellulose fibers can get into the salty water of these salt lakes. The most direct means involves bacteria that live in the water and extrude cellulose fibers. Another means would be the growth and then disruption of algae that live in the water. Finally, one can imagine that trees living in the vicinity of the lake shore would fall into the water and slowly decompose into a solution of cellulose fibers. When we examine water from the modern Great Salt Lake in Utah with Dr. Baxter, it is filled with cellulose fibers just like those we visualized in the ancient salt crystals.

Cellulose is the basic building block of wood and leaves and does not require any chemical processes to release it as single cellulose fibers. Cellulose fibers extruded from bacteria are in this small fibrous form to begin with.

We do not currently know the origin of the DNA we have seen. That will require isolating enough so that we can determine its sequence. This should "spill the beans," so to speak, as to its origin. This remains a high research priority. Smaranda Willcox and I are currently developing the tests needed to read this ancient DNA.

Q: What precautions are taken to ensure the unlikely, but possible, release of some ancient microbe from the salt which could be deadly to the world's population but have no current natural resistance? Billy, Augusta, Georgia

Griffith: Dear Billy,
The best answer here is that nature has been spilling the contents of these salt deposits back into nature for millions of years. While the Salado deposit where we obtained our samples is sequestered 2,000 feet underground, less than 10 miles away the deposit comes close to the surface, and the salt and its contents are being leached back into nature. Hence were there any "andromeda strain" microbes in the salt, we and our ancestors would have been exposed a long time ago. In the high mountains of Asia, there are also ancient deposits where the salt is exposed to the surface. You often see "salt lamps" in stores, and these come from those deposits. Bottom line: not to worry.

Q: How did you feel when you first saw those stringy things under the microscope? Edward, Grade 6, Bellingham, Washington

Griffith: Dear Edward,
We were most excited. It was clearly biological in nature and form, but was something very, very different from anything we had ever seen in the electron microscope. Being very beautiful in their appearance, we took a large number of photographs and made large prints to view them in more detail. It was through a series of tests and exclusions of things that it could not be that we determined it must be cellulose. Comparison with photos of modern cellulose taken by others then confirmed our belief that they were ancient cellulose fibers.

Seeing new things in the electron microscope is one of the most exciting things that we have the honor of being able to do in the laboratory.

Q: Hi Dr. Griffith: What are some of the most surprising/fascinating/unusual things you have seen using electron microscopes? How about in the work of others?

Also, can you suggest links to sites, yours or others', that show particularly interesting EM photographs?

Thanks very much. Wilson P. Stevens, Phoenix, Arizona

Griffith: Dear Wilson,
An analogy: My wife and I have 4 cats, 2 dogs, and 5 horses, so it is like asking which one do we love most? We love them all. Similarly, almost everything I have had the fortune to see in the electron microscope since I began this work in the late 1960s has been exciting. The most exciting things, however, are any molecules or structures that have never been seen by humankind before. Examples were our first visualization of the basic building blocks of chromosomes, called nucleosomes, or even earlier the first visualization of a protein molecule bound to a DNA. More recently, our finding using the electron microscope that the ends of human chromosomes are arranged into giant loops generated not only excitement in the molecular biology community, but a lot of very lovely electron microscope photos. The series of photos that two students in our lab, Mihnea Mangalea and Britta Hackler took of the bacterial viruses (bacteriophages) that they isolated together with Dr. Baxter from the water of the Great Salt Lake have been very exciting since no one had ever seen such viruses from the Great Salt Lake, and the photos are visually very striking. Some of these photos were shown in the program when work on the Salt Lake water was described.

Our website has other examples:

Other examples can be found by a Google search of electron microscope and the topic you are interested in seeing.

Q: Would the cellulose shown in the NOVA program have been parts of much larger plant life, or would the small filaments have constituted all there was? Chabot Community College, Hayward, California

Griffith: Dear Chabot,
Half of the cellulose synthesized on Earth today is made in the oceans by bacteria and algae. The other half is made by land plants. I would thus have to assume that the cellulose we found came mostly from saltwater bacteria and algae, and then to a smaller extent by land plants that had fallen into this large salt lake. Geologists such as Dr. Dennis Powers tell us that it was very arid along the shores of this ancient lake, and this suggests that there may not have been that many trees that would have added their cellulose to the mix in the water. If we can collect enough of this ancient cellulose, it is possible that a method called stable isotope analysis could provide a clue as to whether the cellulose is plant or bacterial in origin.

Q: Was any DNA found with the cellulose, and if so was it possible to relate it to the organism that produced the cellulose? Anonymous

Griffith: Dear Anonymous,
Indeed we have found DNA in this ancient salt, both in extracts from the solid salt and in the tiny liquid inclusions. Until we are able to collect more and work out ways of reading its sequence, we will not be able to tell whether the DNA came from plants, fish, bacteria, or algae. One other common source of DNA would be bacterial viruses (bacteriophages). When we examine modern saltwater from the Great Salt Lake in Utah, we see a lot of DNA, and our belief is that it is from disrupted bacteria and bacteriophages.

Q: In the show, you said when you were going down on the elevator that you were traveling about two million years per second. How does that make you feel knowing that you're traveling back into "deep time" to such a degree? Can you wrap your mind around it?! Mary Johnson, Newark, New Jersey

Griffith: Dear Mary,
One of the greatest pleasures of this project has been the opportunity to spend time around Dr. Dennis Powers, our geologist friend and colleague in the study. Normally at UNC in Chapel Hill, Smaranda Willcox and I spend our days talking to other molecular biologists and seldom have a reason to think in global terms about what we are doing. Driving from El Paso, Texas to the WIPP site with Dennis, one is treated to explanations about each formation, mountain peak, and valley. All this, of course, is couched in terms of millions of years.

With that as a background, when Dr. Nelson ushers us into the tiny, cramped, and pitch-dark elevator (lighted only by our headlamps) and plunge straight downwards into the middle of the Salado formation at a rate of a million years or more per second, one really does have the feeling of going back in geologic time. Of course, when we reach the bottom of the elevator and the door opens into a maze of electric wires, electric golf carts, and telephones, the "Dr. Who"-like fantasy ends abruptly. It returns, however, when we start collecting the ancient salt crystal samples.