The unusually high percentage of silicon-bearing spores in the attack material "is a bit of a strange thing," says Michael. "We have no way of knowing how they were really grown." An anthrax researcher who did not wish to be named calls it "awfully weird" and "a particularly inconvenient exception" because it leaves a gap in the case.Science 19 March 2010:
Vol. 327. no. 5972, p. 1435
Silicon Mystery Endures in Solved Anthrax Case
What about the silicon?
That question has confounded investigators throughout the probe into the 2001 anthrax letter attacks, which the U.S. government formally concluded in February. Scientists inside and outside the government say there is clear evidence that the high levels of silicon found in the anthrax came not from anything added to "weaponize" the anthrax spores—as researchers had suggested early in the probe—but from the culture in which the spores were grown. That evidence may have settled the issue of whether the anthrax was weaponized, at least for scientists familiar with the case. But it raises a different question: Why did the mailed anthrax have such a high proportion of spores with a silicon signature in comparison to most other anthrax samples?
The answer, according to academic scientists who helped with the case, probably would not change the FBI's conclusion that the attacks were the sole handiwork of now-deceased U.S. Army researcher Bruce Ivins. But it could help illuminate exactly how the attack material was prepared. Resolving the mystery might also pave the way for new techniques using trace elements in a bioterrorism agent to link it to its source.
"There's tremendous interest in using metal signatures as a forensic tool," says Adam Driks, an anthrax researcher at Loyola University Chicago in Illinois. But the science to do that is lacking: "We know very, very little about the diversity of elemental composition within spores when they are produced in different ways."
The FBI's scientific case against Ivins rests on DNA tests showing that the mailed anthrax came from a flask under Ivins's control at the U.S. Army Medical Research Institute of Infectious Diseases in Frederick, Maryland. Investigators also had the attack material chemically analyzed, first at the Armed Forces Institute of Pathology (AFIP) in Washington, D.C., within weeks of the attack. Examining the spores under a scanning electron microscope, AFIP scientists detected silicon and oxygen and concluded that the spores had been coated with silica to make them float easily, enhancing their power to kill.
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.
The unusually high percentage of silicon-bearing spores in the attack material "is a bit of a strange thing," says Michael. "We have no way of knowing how they were really grown." An anthrax researcher who did not wish to be named calls it "awfully weird" and "a particularly inconvenient exception" because it leaves a gap in the case. However, neither scientist thinks the anomaly casts doubt on the broader investigation.
The key to the mystery likely lies in the culture medium the perpetrator used to grow the anthrax spores, says Michael. In a recent study, Japanese researchers grew colonies of Bacillus cereus—a close relative of the anthrax bacterium, B. anthracis—in culture media with and without added silicate. Spores grown in the silicate-containing culture showed silicon within the spore coat. In the absence of silicate, there was no silicon, the group reported in January in the Journal of Bacteriology.
One of the study's authors, microbiologist Akio Kuroda of Hiroshima University in Japan, says the precise amount of silicon in individual spores from the anthrax letters could offer clues about the medium. "If the anthrax spores contained a high amount, the suspect must have used a medium that was supplemented with silicon or that intrinsically contained a lot of silicon," Kuroda says. "If a thorough testing of various media sold in the U.S. identifies a few that contain higher amounts of silicon, those could become an investigative clue."