Silicon Analysis of Anthrax Attack Spores: New Answers Leave More Questions Unanswered
We now know conclusively that the spores in the anthrax attacks did not come directly from this flask labeled RMR-1029 by Bruce Ivins, but were cultured most likely using RMR-1029 as the culture inoculum.
Among the many enduring scientific mysteries surrounding the Amerithrax investigation of the anthrax attacks of 2001 is the question of whether an agent such as fumed silica was used to "weapoinze" the spores. Early in the analysis of the attack, statements were issued claiming that the spores were indeed weaponized with silica, but subsequent analysis has proven that not to be the case. New data just released help to pinpoint the reason for the earlier misunderstanding while at the same time emaphasizing that central questions about the conclusion that Bruce Ivins acted alone remain unanswered.
To review, here is a key Washington Post article from October, 2002 that fed into the "weaponized" theory:
A significant number of scientists and biological warfare experts are expressing skepticism about the FBI’s view that a single disgruntled American scientist prepared the spores and mailed the deadly anthrax letters that killed five people last year.
These sources say that making a weaponized aerosol of such sophistication and virulence would require scientific knowledge, technical competence, access to expensive equipment and safety know-how that are probably beyond the capabilities of a lone individual.
Several sources agreed that the most likely way to build the coated spores would be to use the fine glass particles, known generically as "fumed silica" or "solid smoke," and mix them with the spores in a spray dryer.
Interestingly, these speculations in the Post were going well beyond the initial disclosures about silicon. Here are snippets of a White House briefing on October 29, 2001. Major General Parker was the head of USAMRIID, the facility where Ivins worked:
Q: Does that suggest then that there was no additive, there’s been nothing in the spores to make them more — or nothing added to the spores to make them more easily aerosolized?
MAJOR GENERAL PARKER: Complicated question. We do know that we found silica in the samples. Now, we don’t know what that motive would be, or why it would be there, or anything. But there is silica in the samples. And that led us to be absolutely sure that there was no aluminum in the sample, because the combination of a silicate, plus aluminum, is sort of the major ingredients of bentonite.
However, the problem is that even though the element silicon was found to be present at higher than expected concentrations in the attack material, the spores were not "coated". Here is a key disclosure of the lack of coating:
Harvard University molecular biologist Matthew S. Meselson, who has consulted for the FBI on the anthrax probe, dismisses these early statements as misunderstandings or misinterpretations of the scientific studies conducted on the Daschle powder. "I don’t know of anybody with spore expertise who actually worked on the stuff who said the spores were coated," he says. The FBI has never publicly claimed the spores were coated with silica and, in fact, told members of Congress at classified briefings that the spores were not coated, he says.
Meselson, who reviewed Beecher’s article for the FBI, was asked to assess scanning electron micrographs of the anthrax powder. Early in 2002, he spent half a day at the FBI’s Washington field office and looked at "a large heap of electron micrographs" of the powder from the Daschle letter.
"I saw no evidence of anything except spores, no evidence of silica nanoparticles," Meselson says. "If silica was present, I would have seen it, but nothing could have been purer than what I saw," he insists. Though purified, the preparation "had not been milled," he adds.
I have to note here, that as a Ph.D molecular biologist, the opinion of Mesleson carries tremendous weight for me. Meselson began his career by publishing what later came to be called "The Most Beautiful Experiment in Biology". [To appreciate the elegance of the experiment, see this WikiMedia Commons illustration and watch this brief animation.]
While still under the impression that an agent such as fumed silica was used to weaponize the attack spores, work was carried out at Dugway in attempt to produce similar material. The Chemical and Engineering News article linked above summarizes some of that work and the results. Other parts of the work, including electron micrographs of coated and uncoated spores of a similar bacterial species were published here.
The newest information about the silicon mystery comes in a news article in the March 19 issue of the journal Science. Here, we see conclusive visual evidence that the silicon (in green in the upper frame) in the attack spores is located inside the outer covering of the spores:
Sandia National Laboratories illustration as reproduced in Science
Here is how the article described the analysis carried out at Sandia National Laboratories:
A more detailed analysis by Joseph Michael and Paul Kotula of Sandia National Laboratories in Albuquerque, New Mexico, contradicted that conclusion. Studying individual spores with a transmission electron microscope, they found that the silicon was located within the spore coat, well inside the cell’s exosporium (outermost covering). By contrast, when they looked at surrogate spores weaponized with silica, the silicon was clearly outside the exosporium.
But the Sandia study, presented last September to a National Academies panel reviewing the science behind the investigation, still leaves questions. Out of 124 spores from a letter mailed to Senator Patrick Leahy of Vermont, Michael found the silicon-and oxygen signature in 97—78% of the sample. The signature was present in 66% of a sample from a letter to former Senator Tom Daschle and in 65% of spores from a letter sent to the New York Post.
Out of nearly 200 other anthrax samples from different labs, none came close to displaying such a prominent silicon signature. The highest, in a sample from Dugway Proving Ground in Utah, was 29%. The researchers couldn’t find silicon in the coat of a single spore out of some 300 taken from RMR-1029, the flask in Ivins’s lab identified as the source of the bacteria used in the attacks; they concluded that all the silicon had come from the culture.
These results show conclusively that the silicon content of the attack spores differed from the silicon content of the spores that were in the actual RMR-1029 flask pictured above. The results suggest that the silicon inside the spores used in the attack came from the culture medium in which the spores were grown. Researchers in Japan recently published results with the bacterium Bacillus cereus that provide further support for this interpretation.
These researchers grew B. cereus cultures on growth medium containing high or low concentrations of silicates and found that spores produced after growth on the high silicate medium had a layer of silicon inside the spore that looked like the illustration above for the anthrax spores used in the attacks. They then carried out various experiments on high and low silicon spores to determine how the high silicon spores differed from the low silicon spores. They found no difference in spore dispersion in air (although I have reservations about whether they first achieved the same level of purification away from debris that is reported for the attack material) but did find the high silicon spores were more resistant to disruption in acid, a trait that could have evolutionary advantages. Their final sentence of the publication is the most important here:
Our findings also strongly indicate that the anthrax spores were harvested from culture on a silicate-containing medium.
The published findings of the Amerithrax investigation suggest that detailed genetic analysis (that has not yet been released in full) indicates that the attack spores share a unique subpopulation of four mutant strains along with the primary Ames strain that was only found in Ivins’ RMR-1029 flask or cultures that were known to be directly derived from it. These newly released silicon results conclusively show that the attack spores could not have been taken directly from the RMR-1029 flask, but would have to have been cultured on a high silicate growth medium. In this diary, I showed that the amount of spore material that was present in the attack letters would have been very difficult for Ivins to culture on his own in the small shake flasks he had available to him. Instead, it would have been much easier for a researcher with access to a fermenter to produce such a large amount of spores. In this diary, I pointed to a facility at Dugway that has a fermenter of just the right size to produce the material in only one or two batches. Isn’t it interesting that the second highest silicon content batch of spores analyzed by Sandia came from Dugway?