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FBI Ignored, Hid Data Potentially Excluding Bruce Ivins as Anthrax Killer

9:16 am in Uncategorized by Jim White

FBI photos of the material in the letter sent to Senator Pat Leahy (left) and to the New York Post (right), from the report.

A report from McClatchy provides important new evidence and analysis in the FBI’s Amerithrax investigation of the 2001 anthrax attacks. The report shows that the FBI ignored as potentially erroneous a measurement of silicon in one anthrax sample and then hid this information from Congressman Jerrold Nadler (D-NY). Even more importantly, the high silicon measurements in at least two samples also were coupled with high tin measurements, opening up the possibility that silicon was added to the attack material in a form that is not mentioned in any of the FBI documents. Significantly, it is virtually impossible that Bruce Ivins, whom the FBI has concluded acted on his own to carry out the attacks, would have been able to perform the necessary chemical manipulations involved in this treatment of the spores.  Ivins likely also would not have had access to the necessary laboratory equipment to perform this treatment.

The presence of silicon and how it may have gotten into the anthrax material has been a point of great controversy throughout the entire investigation. This question is important because the chemical nature of the silicon and the level at which it is present is presumed to be an indicator of whether the anthrax spores have been “weaponized” to make them suspend more readily in air so that they are more effective in getting into the small passageways of the lungs of the intended targets of the attack. Early in the investigation, Brian Ross published “leaked” information that the spores had been weaponized through addition of bentonite and that Iraq had a weaponization program that used bentonite. This report turned out to be false, as no evidence for bentonite has been found. A more sophisticated type of weaponizing would rely on mixing the spores with nanoparticles of silica (silica is the common name for the compound silicon dioxide) to make them disperse more easily.

The FBI carried out a special form electron microscopy that could identify the location of the silicon in the spores from the attack material. They found that the silicon was in a structure called the the spore coat, which is inside the most outer covering of the spore called the exosporium. If silica nanoparticles had been used to disperse the spores, these would have been found on the outside of the exosporuim (see this diary for a discussion of this point and quotes from the scientific literature) because they are too large to penetrate it.  No silicon signature was seen on the outside edge of the exosporium.  What is significant about the type of silicon treatment suggested in the McClatchy piece is that both high silicon and high tin measurements were found in several samples and that there is an alternative silicon treatment that would involve a tin-catalyzed polymerization of silicon-containing precursor molecules. McClatchy interviewed scientists who work with this process and they confirmed that the ratio of silicon to tin found by the FBI is in the range one would expect if such a polymerization process had been used. Read the rest of this entry →

McClatchy Points Out Key FBI Failure in Amerithrax Investigation

5:58 am in Uncategorized by Jim White

The infamous RMR-1029 flask. Where is the B. subtilis flask?

In an article published Wednesday evening on their website, McClatchy points out yet another failing in the FBI’s Amerithrax investigation of the 2001 anthrax attacks that killed five people. The article focuses on the fact that the FBI was able to get a clear genetic fingerprint of a bacterial contaminant that was found in the attack material mailed to the New York Post and to Tom Brokaw (but not to either Senator Daschle or Senator Leahy). This contaminant, Bacillus subtilis, is used in some cases by weapons laboratories as an anthrax simulant, because its behavior in culture and in drying the spores is very similar to Bacillus anthracis but it is easier to handle because it is not pathogenic.  I covered the FBI’s failure to link this B. subtilis contaminant to Ivins in this diary in February of 2010.

A key finding in the McClatchy article comes from an FBI memo they obtained:

One senior FBI official wrote in March 2007, in a recently declassified memo, that the potential clue “may be the most resolving signature found in the evidence to date.”

The genetic fingerprint of the B. subtilis contaminant, in other words, was a very definitive clue that needed to be traced to the culprit. The problem, as McClatchy points out, is that the FBI never got a match to materials from Ivins or from anyone else:

Yet once FBI agents concluded that the likely culprit was Bruce Ivins — a mentally troubled, but widely regarded Army microbiologist — they stopped looking for the contaminant, after testing only a few work spaces of the scores of researchers using the anthrax strain found in the letters. They quit searching, despite finding no traces of the substance in hundreds of environmental samples from Ivins’ lab, office, car and home.

LSU anthrax researcher Martin Hugh-Jones makes the key point in his discussion with McClatchy, in response to being informed the government tested thousands of samples for a match to the B. subtilis contaminant, “But were they thousands of the right samples?”

The significance of B. subtilis being present in some of the attack material cannot be overstated, especially when the FBI admits that they were able to obtain a unique DNA fingerprint of the strain that was present. As I wrote last year, I suspect that Defense Department personnel at either Dugway or Batelle cultured the material used in the attacks and noted personnel there have worked with both anthrax and B. sublitlis:

Even though Ivins can’t be linked to the particular B. subtilis strain used, there is a documented case of B. subtilis being used as a B. anthracis simulant at another facility where we already know that much of the material that went into RMR-1029 was produced. Recall from this diary that I analyzed the available information about the amount of B. anthracis used in the attacks and found it highly unlikely that Ivins could have cultured the large amount of spores used in the attacks with the equipment and time he had available. Much of the material in RMR-1029 was produced at Dugway.

On December 13, 2001, Judith Miller published an article in the New York Times, where she disclosed that “government officials have acknowledged that Army scientists in recent years have made anthrax in a powdered form that could be used as a weapon.” She further pointed out that this work occurred at Dugway in 1998. It should be noted that the anthrax produced at Dugway for Ivins that went into RMR-1029 was cultured in 1997.

The Miller article then goes on to quote scientist William C. Patrick on how he coached scientists at Dugway in drying a pound of highly purified anthrax spores in 1998. Miller quotes a Dugway spokesperson as saying a strain different from Ames (the parent of RMR-1029) was used in the drying experiments. Note that a pound of spores is enough dried spores to produce hundreds of letters with the one to two grams of dried spores thought to be in each letter. But in a further bit from the Dugway spokesperson, we have this:

She said Dugway did make one- pound quantities of Bacillus subtilis, a benign germ sometimes used to simulate anthrax.

We know that the key anthrax work at Dugway and Batelle was carried out by the Defense Intelligence Agency. I find it highly unlikely that this agency would be completely forthcoming with the FBI in sharing samples of all of the B. anthracis or B. subtilis strains in its collection, so Hugh-Jones was right to question whether the FBI tested the right samples for a match to the key B. subtilis contaminant. The fact that all of Ivins’ materials were available to the FBI, and that the FBI was able to carry out extensive environmental sampling (albeit several years after the preparation of the attack materials) where Ivins worked and lived comes very close to excluding Ivins as a suspect since the contaminant was not found in association with him.

Buried in the McClatchy article is an admission from a source close to the investigation that seems to shed some light on why the FBI continues to insist that Ivins was the sole attacker, even going so far as to concoct an after-the-fact “forensic psychiatric profile” in an effort to bolster their case:

“If they ever had any doubts, once he committed suicide, they had to unite,” this person said. “Otherwise, you’ve driven an innocent man to suicide. And that’s a terrible thing.”

Yes, driving a man to suicide is a terrible thing. But is it any less terrible to continue to insist on the guilt of a man who cannot conclusively be proven to have carried out these horrendous attacks ten years ago?

Rush Holt on NAS Anthrax Report: FBI Has Not Proven Open and Shut Case

1:41 pm in Uncategorized by Jim White

Rep. Rush Holt (D-NJ)

Congressman Rush Holt (D-NJ), from whose district several of the letters were mailed in the 2001 anthrax attacks, is concerned about the issues raised in the National Academy of Sciences report on its analysis of the science behind the FBI’s investigation. See this post from earlier today, where I raise the question of whether the NAS report would have created reasonable doubt for a jury evaluating the case against Bruce Ivins had he survived. Congressman Holt also would appear to doubt the FBI’s conclusions, as the release below notes that “The FBI has not proven to me that this is an open and shut case.”

Noting that “There are still questions to be answered”, Congressman Holt just released the following, in which he calls for a Congressional Anthrax Commission:

FOLLOWING NATIONAL ACADEMY REPORT, HOLT CALLS FOR CONGRESSIONAL ANTHRAX COMMISSION

“NAS Report Makes Clear There Are Still Questions to be Answered and Still Lessons to be Learned”

(Washington, D.C.) – Rep. Rush Holt (NJ-12) today is reintroducing the Anthrax Attacks Investigation Act, legislation that would establish a Congressional commission to investigate the 2001 anthrax attacks and the federal government’s response to and investigation of the attacks. Holt is introducing the bill on the same day that the National Academy of Sciences issued its report raising questions about the Federal Bureau of Investigation’s (FBI) scientific conclusions in the “Amerithrax” case. He first introduced the legislation in September 2008.

“The NAS report makes clear there are still questions to be answered and still lessons to be learned about the FBI’s investigation into the attacks,” Holt said. “It would take a credulous person to believe the circumstantial evidence that the FBI used to draw its conclusions with such certainty. The FBI has not proven to me that this is an open and shut case. We still badly need a 9/11-style commission to determine how the attacks happened and whether we learned the lessons to prepare for another attack.”

The 11-member bipartisan commission would investigate the attacks, assess the federal government’s response to and investigation of the attacks, and make recommendations to the President and Congress on how the country can best prevent or respond to a future bioterror attack. Just as the 9/11 Commission looked not only at the attacks of that morning, but also at recommended changes in the structure of government agencies, screening methods, and Congressional oversight, an anthrax commission would look not only at the attacks, but also measures for prevention, detection, and investigation of any future bio-terrorism.

The commission would consider scientific, technical evidence as well as classified evidence the NAS did not examine.

The 2001 attacks evidently originated from a postal box in Holt’s Central New Jersey congressional district, disrupting the lives and livelihoods of many of his constituents. Holt has consistently raised questions about the federal investigation into the attacks.

“Too many questions remain about the anthrax attacks and the government’s bungled response to the attacks,” Holt said. “A high level commission, like the 9/11 Commission, would be a start-to-finish examination of the many outstanding questions, and it would help American families know that the government is prepared to protect them and their children from future bioterrorism attacks.”

I have requested a copy of the bill from Congressman Holt’s office and will post it as an update.

Update: The bill can be read here: HOLT_009_xml (pdf).

Would the NAS Report Have Led to Reasonable Doubt in an Ivins Trial?

12:02 pm in Uncategorized by Jim White

FBI photos of the material in the letter sent to Senator Pat Leahy (left) and to the New York Post (right), from the report.

It seems very likely to me that had Bruce Ivins not died, the analysis carried out by a panel from the National Academy of Sciences in assessing the scientific evidence tying Ivins to the 2001 anthrax attacks would have led to reasonable doubt on whether Ivins carried out the attacks. For this post, let us concentrate only on the NAS response to FBI claims on the spores used in the attack, especially with regard to how the spores were prepared.

What we do know from the report is that the spores used in the attacks did not come directly from Bruce Ivins’ RMR-1029 flask, but had to undergo a culturing step if RMR-1029 was even the source. Also, the spore material in different individual mailings differed in purity and particle size.  Silicon was present in the spores, but was not added as a step to “weaponize” the spores.  Instead, the silicon was incorporated into the coating of the spores themselves.   Finally, the NAS panel did not feel there was sufficient evidence to support the FBI claim that a highly skilled person had prepared the material.

Overall, the importance of the primary conclusion of the NAS report cannot be overstated (p. 4 of the report as marked, all references will use internal page numbers, not pdf numbers from my pre-publication copy):

It is not possible to reach a definitive conclusion about the origins of the B. anthracis in the mailings based on the available scientific evidence alone.

A good defense attorney probably would need no more than that conclusion to establish reasonable doubt in a trial.  But the details on how the panel reached that conclusion are important.

In this post, I discussed whether Ivins could have produced all of the material used in the attacks with the equipment he had available and without drawing attention to himself.  That post opened with a discussion of how many spores were known to have been present in the attacks.  I looked at what was in the FBI’s report, filled in some gaps with my experience in microbiology and came to the conclusion that somewhere between four and seventy liters of liquid culture could have produced the attack material.  The NAS report comes to a similar conclusion on page 62:

Thus, cultivation in the range of 2.8 to 53 liters of liquid medium would have been required to produce the spores required for the letters (see Table 4.2).

Interestingly, the panel also calculated that it would have required between 463 and 1250 agar plates if the attack material had been produced on solid medium.  It seems highly unlikely Ivins could have cultured this many extra Petri dishes without someone in the lab taking note and reporting it to the FBI once the investigation began.

The photo above shows how dramatically different the highly purified material in the letter sent to Senator Patrick Leahy’s office was when compared to the material mailed to the New York Post.

There had been much speculation early on in the press that the material sent to Senator Leahy’s office had been “weaponized” by the addition of materials including silicates to make tiny particles remain suspended in the air so that they could be inhaled.  The report documents that although silicon is found in the spores used in the attacks, the silicon is localized in the spore coats.  This point is driven home very clearly in this photo, where the silicon can be seen “lighting up” within the outer lining of the spores:

Many experiments were carried out in an attempt to match the silicon content, the particle size distribution and the degree to which the final material would remain suspended in air.  I will rely here on the entire summary the panel provided on the issue of silicon in the spores (page 71):

The substantial effort devoted to the characterization of silicon in Bacillus spore coats resulted in new fundamental insight into microbial processes and the development of new or enhanced analytical measurement technology. (Table 4.4 presents a summary of the analytical results.) Elemental analysis of the letter samples showed that 1) the silicon content was high, 2) most of the silicon was incorporated in the spore coat, 3) the majority of spores in the samples contained silicon in the coat, and 4) no silicon was detected in the form of a dispersant in the exosporium.

The bulk silicon content in the Leahy letter could be completely explained by the amount of silicon incorporated in the spores during growth. (Not enough material was available to make this comparison for the Daschle letter.) In contrast, the New York Post letter had significant bulk silicon content, far exceeding that contained in the spores.

No studies have considered the effect of the chemical form of silicon (e.g., silicate impurity versus polydimethylsiloxane antifoam agent) on uptake. The inability of laboratory experiments to reproduce the silicon characteristics of the letter samples is not surprising given the complexity of the uptake mechanism.

A few spores analyzed from RMR-1030 contained silicon in the coat, but none of the spores analyzed from RMR-1029 contained silicon in the coat. Therefore, the letter samples could not have been taken directly from the flasks—a separate growth preparation would have been required.

The material in the Daschle and Leahy letters was reported to have “a high level of purity” and to have electrostatic properties that caused it to disperse readily upon opening of the letters. These properties should be regarded as qualitative observations since they were not based on quantitative physical measurements. The committee received testimony (Martin, 2010) stating that some Dugway preparations, particularly those utilizing lyophilization but no dispersant, gave products with similar appearance and electrostatic dispersibility as the letter samples, suggesting that these properties were not necessarily connected to an intentional effort to increase dispersibility through addition of a dispersant. Exogenous silicon and bentonite, which enhance the dispersibility of spore preparations, were not found in the Leahy and Daschle letters.

Note that this analysis provides the strongest evidence to date that the spores used in the attacks did not come directly from the RMR-1029 flask, because the spores in the flask do not have the silicon content as those in the attack material. Note also that growth in the presence of polydimethylsiloxane-containing antifoam agents is seen as one route that needs to be investigated for how silicon can be incorporated at elevated concentration in the spores, just as I suggested in this post.

It is also worth noting that highly purified spores produced at Dugway did have the aerosolizing quality of the attack material. No additional treatments besides purification were needed for these spores to disperse in air, so I suspect that is one of the primary reasons that the NAS panel could not support the FBI claim that someone with a very high level of expertise must have prepared the material used in the attacks.

National Academies on FBI Anthrax Investigation: Scientific Link to RMR-1029 Not as Definitive as DOJ Claim

8:00 am in Uncategorized by Jim White

The infamous RMR-1029 flask.

With a live-streaming press briefing, the National Academies is releasing the report prepared by a committee from the National Academy of Sciences that has reviewed the scientific portion of the FBI’s Amerithrax investigation into the anthrax attacks of 2001. The report is meant to analyze only the science involved in the FBI’s investigation.

Key findings, from the summary:

It is not possible to reach a definitive conclusion about the origins of the B. anthracis in the mailings based on the available scientific evidence alone.

The FBI created a repository of Ames strain B. anthracis samples and performed experiments to determine relationships among the letter materials and the repository samples. The scientific link between the letter material and flask number RMR-1029 is not as conclusive as stated in the DOJ Investigative Summary.

Silicon was present in the letter powders but there was no evidence of intentional addition of silicon-based dispersants.

On first glance, this report appears to be devastating to the scientific conclusions drawn by the FBI in their investigations. Another key finding:

It is difficult to draw conclusions about the amount of time needed to prepare the spore material or the skill set required of the perpetrator.

I will have more detail on the report as I have time to read it. In the meantime, previous posts I have written on the science include this one on whether Ivins could have produced all the material (relating to the excerpt just above) and this report on the presence of silicon.

Rush Holt Blasts FBI for Withholding Documents from Outside Review of Scientific Work in Anthrax Investigation

5:07 am in Uncategorized by Jim White

The infamous RMR-1029 flask genetically linked to the anthrax attack material.

[Ed. note: Link to Rep. Holt's statement and letter have been added below.]

Both the New York Times and McClatchy report that Congressman Rush Holt (D-NJ) has written a letter to the FBI, blasting them for requesting a delay in the release of the final report from the National Academy of Sciences panel that has been reviewing the scientific analyses used in the FBI’s Amerithrax Investigation of the 2001 Anthrax mailings. It appears that in requesting the National Academies to delay release of its final report, the FBI has released an additional 500 pages of documents to the panel, but only after having seen the draft final report from the panel.

Here is McClatchy discussing Holt’s letter (which I don’t see posted on Holt’s website; I will call and request a copy Link to PDF letter here):

Holt, a scientist and the chairman of the House Select Intelligence Oversight Panel, said the academy recently shared with the bureau its draft report on the “Amerithrax” investigation, a narrow scientific review that the FBI requested in 2008 in an effort to quell controversy over its findings that a disgruntled government scientist was behind the attacks.

“This week I was informed by the NAS that the FBI would be releasing an additional 500 pages of previously undisclosed investigative material from the Amerithrax investigation to the NAS,” he wrote. Holt said he understands that the “document dump . . . is intended to contest and challenge the independent NAS panel’s draft findings.”

“If these new documents were relevant to the NAS’ review, why were they previously undisclosed and withheld?” Holt wrote. He requested a meeting with the FBI director.

In the Times, Scott Shane reports that the National Academy has agreed to extend its study:

E. William Colglazier, the academy’s executive officer, said the F.B.I.’s request was a surprise and came after the bureau saw the panel’s peer-reviewed final report, which was scheduled for release in November. He said that the committee’s 15 members, top scientists who serve as volunteers, were “exhausted,” but that the panel had agreed to extend the study and consider revising the report in return for an additional fee, probably about $50,000, beyond the $879,550 the F.B.I. has already paid for the study.

Dr. Colglazier declined to say if the report was critical of the F.B.I.’s work but said it was “very direct.” The report sticks to science and does not offer an opinion on whether Dr. Ivins carried out the anthrax attacks, he said.

The McClatchy article also quotes Holt as saying of the FBI that it “consistently botched and bungled this case from the beginning.” In addition to the early focus on Steven Hatfill as the primary suspect, followed by a settlement of more than $4 million paid to Hatfill after he was cleared of involvement, several aspects of the FBI case do not appear to withstand scientific scrutiny.

In this diary, I pointed out that the amount of highly purified anthrax material that was used in the mailings would have been very difficult, if not impossible for Bruce Ivins, whom the FBI stated was solely responsible for the attacks, to have produced at his government laboratory without arousing the suspicion of his coworkers. The small shake flasks that Ivins would have used produce very little material, so he would have had to grow anthrax in a very large number (over 35 or so) of the cultures he normally grew.

On the other hand, a single production of spores from a fermenter of at least 70 liters would have produced enough anthrax spore material to account for what was used in the attacks. Further, in this diary, I point out that the abnormal silicon content of the spores used in the attack can be accounted for by the presence of an agent called “antifoam”, that is added to microbial cultures when they are grown in large fermenters, again suggesting that the attack material was produced in a fermenter to which Ivins did not have access.

I had been monitoring the website for the National Academies investigation regularly since the project’s slated termination in late October, looking for their final report. It appears now that we all will have to wait a bit longer before we see that report. My only hope is that Rush Holt is making sure that David Margolis is not allowed anywhere near the report before it is made final.

(h/t to @jaraparilla for alerting me to the Times article and to Retired Military Patriot for finding the McClatchy article)

Hatfill Speaks Out: “You Can’t Turn Laws On and Off As You Deem Fit”

6:46 am in Uncategorized by Jim White

On Friday, Steven Hatfill appeared on NBC in an interview with Matt Lauer. This was Hatfill’s first public appearance after being cleared as a suspect in the 2001 anthrax attacks and after receiving a multi-million dollar settlement from the government. The full interview is compelling.

At around six minutes in the video, we have this from Hatfill, as transcribed in the accompanying article:

“I love my country,” Hatfill, 56, told Lauer. But, he added, “I learned a couple things. The government can do to you whatever they want. They can break the laws, federal laws, as they see fit … You can’t turn laws on and off as you deem fit. And the Privacy Act laws were put in place specifically to stop what happened to me. Whether we’re at war or have been attacked, the foundation of society is that you hold to the laws in place. I used to be somebody that trusted the government. Now I really don’t trust anything.”

An extended article by David Freed drawing on additional interviews with Hatfill appears in The Atlantic .

Hatfill repeats his statement about the government being able to do whatever it wants, but this time in the context of a particularly disturbing episode when the FBI was hounding him:

Boo was driving Hatfill to a paint store a week later when FBI agents in a Dodge Durango, trying to keep up with them, blew through a red light in a school zone with children present. Hatfill says he got out of his car to snap a photo of the offending agents and give them a piece of his mind. The Durango sped away—running over his right foot. Hatfill declined an ambulance ride to the hospital; unemployed, he had no medical insurance. When Washington police arrived, they issued him a ticket for “walking to create a hazard.” The infraction carried a $5 fine. Hatfill would contest the ticket in court and lose. The agent who ran over his foot was never charged.

“People think they’re free in this country,” Hatfill says. “Don’t kid yourself. This is a police state. The government can pretty much do whatever it wants.”

As Freed probes the merciless way in which the FBI hounded Hatfill, he eventually gets to this exchange with him:

The next morning, driving through Georgetown on the way to visit one of his friends in suburban Maryland, I ask Hatfill how close he came to suicide. The muscles in his jaw tighten.

“That was never an option,” Hatfill says, staring straight ahead. “If I would’ve killed myself, I would’ve been automatically judged by the press and the FBI to be guilty.”

Having failed to close the case in such a way with Hatfill, the FBI did achieve precisely that closure with Bruce Ivins.

Hatfill’s accusation that the government turns laws on and off as it sees fit applies to far more than the privacy laws in his case. Our government has turned off its laws against torture. It has turned off its laws against illegal wiretapping. It has turned off its laws against financial fraud by the largest corporations. Who will switch these laws back on?

Most Likely Source of Silicon in Anthrax Attack Spores Argues Against Production by Ivins

9:16 am in Uncategorized by Jim White

In "Silicon Analysis of Anthrax Attack Spores: New Answers Leave More Questions Unanswered", I referred to data recently published in Science, where it was found that the anthrax spores used in the attacks of 2001 contained an unexpectedly high concentration of silicon inside them, as a component of the internal spore coat. I also discussed data from a paper by researchers in Japan who demonstrated that they could produce spores with high silicon content using a closely related bacterium by culturing the bacteria in medium containing high concentrations of silicates.

I am deeply indebted to commenter behindthefall (see this comment as just one in the series) in the comments section of the diary linked above for continuing to ask why someone would put a high concentration of silicates in the growth medium. Those persistent "why?" questions kept coming at me, and I finally extended my thinking from just the narrow question of silicates in the medium to think more broadly about any material containing the element silicon which could somehow wind up in the spores. That took me directly to materials called antifoam agents.

Before getting to the silicon content of popular antifoam agents, a brief digression to explain foaming in microbial cultures is necessary. When microbial fermentation is carried out in large fermenters as opposed to small shake flasks, it is common practice to add agents generally classed as antifoams. Microbial growth rate in liquid medium is often limited by the rate of oxygen transfer into the medium. In shake flasks, the flask is filled below the half-way mark and oxygen is supplied simply by swirling the flask with it attached to a moving platform. Oxygen transfer occurs at the liquid-air interface and keeping the liquid circulating in this way allows oxygen to achieve a sufficient concentration in the liquid to support growth. In larger fermenters, on the other hand, the liquid is much "deeper" and so must be both stirred with a mechanical stirrer and aerated through the use of forced air generally introduced at the bottom of the tank, similar to the air pumps commonly used in aquariums.

To appreciate the foam problem that forced aeration induces, consider two different glasses containing a carbonated soft drink. First, consider a highball glass (for you non-drinkers a higball glass ironically has a low profile, just taller than the height of an adult hand and with a similar diameter) filled less than halfway. Swirling this glass gently by hand isn’t going to cause much trouble for containing the liquid. That is the situation seen in shake flask cultures. Now consider a much taller glass tumbler with a narrow diameter and filled to about the three-quarters mark. Imagine that the soft drink is being stirred by a small propeller and you then insert a straw to the bottom of the glass and blow. That is the foamy mess encountered in large fermenters if steps are not taken to control foam.

Antifoam agents work to reduce the surface tension on bubbles, collapsing them.

Although there are multiple types of antifoam agents employed in microbial fermentation, silicone based antifoams are among the most popular. My favorite antifoam agent of all time is Dow Corning Antifoam M (pdf) because in addition to its use in fermentation, it also is used as an antiflatulent.

Here are the typical properties of this material from the Dow website I linked above:

Antifoam M properties

From a biochemical perspective, it seems quite unlikely that the dimeticone itself (polydimethylsiloxane is a large, polymeric molecule with lots of silicon in it) would be able to be taken up by anthrax cells in culture. However, the presence of four to seven percent of the material as silica is quite intriguing, because very small particles of silica carried in the mixture of silicon polymer could be expected to be available for movement into the cells. (In the calculations that follow, I will assume a silica content of 5%.)

That thought prompted a return to the paper from the Japanese researchers to look again at what they had to say about the chemical structure of the silicon they found in spores. It turns out that although they supplied silicon to the cultures in the form of silicates, the silicon inside the spores was most likely present as silica (the "HF" they refer to is hydrofluoric acid, which is a very strong acid that is different from the other "mineral acids" to which the high silicon spores are resistant):

As far as we know, diatoms, plants, and animals accumulate silicate as silica (13). Silica can be dissolved in HF (16). Accordingly, if the Si layer of spores contains silica, it could be removed from the high-Si spores with HF treatment. Approximately 75% of Si that was accumulated in the spores was released as silicate after treatment with 50 mM HF (data not shown). We compared the acid resistance of HF-treated high Si- and low-Si spores (Fig. 7). After HF treatment, the viability of the high-Si spores was no longer higher than that of the low-Si spores. These results indicated that the Si layer mainly contains silica and supports acid resistance.

It seems very likely to me that anthrax grown in the presence of antifoam agents that contain silica would be able to incorporate this silica directly into the spore coat, skipping the step of converting silicates to silica. It appears that typical working concentrations of antifoam agents could achieve silica concentrations in the range at which silicates were incorporated into medium in the experiments in Japan. The silicate concentration in their experiments was 100 micrograms of silicate per milliliter of culture medium. That corresponds to roughly 0.01% of the medium’s total weight in silicates.

Antifoam agents can be effective at very low concentrations. For example, see here for a recommendation for use at 0.005 to 0.02% for the polymer, so for Antifoam M the silica would be only at 0.001% of the weight of the medium, 10-fold lower than the silicate concentration fed in the reported experiments. However, it is common to exceed those low recommended levels. For example, see this publication (pdf) from 1973, where a silicone antifoam was added to a final concentration of 0.5%. In this case, if the agent were Antifoam M, the silica concentration would be 0.025%, well above the 0.01% silicate fed in the experiments in Japan. Also, my own personal experience running a fermentation pilot plant involved many fermentation runs I can recall that added up to a full one percent or more of the total medium volume as polymeric antifoam before the process ended.

If the silicon in the anthrax attack spores does indeed come from the material having been cultured in the presence of a silicone antifoam agent that also had silica present, then the FBI’s conclusion that Bruce Ivins acted alone in the attacks is called into serious doubt. In this diary, I calculated that Ivins would have to have grown 36 of his two liter shake flask cultures to produce the spores used in the attacks. I further quoted pages 26 and 27 of the FBI’s Amerithrax Investigative Summary (pdf):

In 1997, USAMRIID commissioned another Army research facility, Dugway, to prepare large batches of Bacillus anthracis spores for an upcoming series of studies testing the anthrax vaccine, because USAMRIID lacked the capacity to do so. By the fall of 1997, Dr. Ivins received from Dugway seven shipments containing the concentrated product of 12 ten-liter, fermenter-grown lots of Bacillus anthracis – the “Dugway Spores.” By Dr. Ivins’s own account, these spores were not in perfect shape, so he had to “clean them up.” Indeed, he even discarded the seventh shipment because he deemed it to be inadequate. He noted in his lab notebooks the process that he used to clean them, and also sent e-mails to various people noting his frustration that he had to wash them. To the Dugway Spores, Dr. Ivins added concentrates of 22 two-liter batches of spores which he himself prepared with the help of a laboratory technician. He combined his spores with those from Dugway, and put them in two flasks, labeled “GLP [Good Laboratory Practices] Spores.” In addition, he created a Reference Material Receipt record on which he made the following notation: “Dugway Proving Ground + USAMRIID Bact’D – highly purified, 95% unclumped, single refractile spores.” Finally, in his laboratory notebook 4010, page 074, he described the end-product of these efforts as “RMR-1029: :99% refractile spores;

The alternative explanation to Ivins growing 36 two liter cultures is one fermenter run of approximately 70 liters or more. Note that the FBI investigative summary informs us that Dugway was engaged for the 1997 work precisely because Ivins did not have access to large scale culture equipment. The fact that the RMR-1029 spores themselves did not have a high silicon content could be explained by the use an antifoam agent that did not have silica present for those particular fermenter runs at Dugway, since silica is not uniformly found in all antifoam agents. However, the presence of high silicon in the attack spores strongly suggests that they could have been grown in the presence of an antifoam agent that did contain silica. If Ivins had grown the spores in his shake flask equipment, he would have had no reason to include any sort of antifoam agent, much less one containing silica, because antifoam is just not used in shake flasks. It also seems unlikely that Ivins would have changed his culture process to produce the attack material. If he did not introduce silicon in his early shake flask cultures (and we know he didn’t from the silicon analysis of the RMR-1029 material), it seems unlikely he would have done so with shake flasks for the attack material.

Note also from the Science report that the only other elevated (but not as high as the attack spores) silicon content spores analyzed came from Dugway, where we know that fermenters are available.

In conclusion, the finding of high silicon in the spores used in the anthrax attacks suggests that these spores were grown in a large fermenter that used an antifoam agent containing silica. Since Bruce Ivins did not have access to a large fermenter, fermenter growth would suggest that he could not have acted alone in the attacks.

This hypothesis could be tested easily in a series of experiments where B. anthracis or B. cereus is grown in media with a range of concentrations of antifoam agents with and without silica present in them. followed by analysis of the silica content of the spores. From an investigation standpoint, it would not be difficult to determine if Ivins or someone in his laboratory ever purchased an antifoam agent containing silica that could have inexplicably been used in shake flasks.

Update: Due to the ongoing conversation in the comments below, it is useful to see the analysis of silicon locations in the high silicon spores in the Japanese study cited above. In the illustration below, CX stands for cortex, CT for coat, SX for particles containing silicon, EX for exosporium and UC for undercoat:

Japanese high silicon spore

To my eye, this silicon coating of the spore coat looks just as contiguous as that in the electron micrographs of the attack spores in the previous diary.

Silicon Analysis of Anthrax Attack Spores: New Answers Leave More Questions Unanswered

1:00 pm in Uncategorized by Jim White

RMR-1029 Flask

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.

/snip/

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.

Read the rest of this entry →

Not So Subtilis: The Missing Contaminant in the Amerithrax Investigative Summary

8:56 am in Uncategorized by Jim White

The Investigative Summary (pdf) published by the FBI in closing the Amerithrax investigation into the anthrax attacks of 2001 is curiously silent on the presence of a second species of bacteria found in the New York Post and Brokaw letters.

One of the search warrant affidavits (page 5 of linked pdf) released previously had this to say about the presence of Bacillus subtilis:

Both of the anthrax spore powders recovered from the Post and Brokaw letters contain low levels of a bacterial contaminant identified as a strain of Bacillus subtilis. The Bacillus subtilis contaminant has not been detected in the anthrax spore powders recovered from the envelopes mailed to either Senator Leahy or Senator Daschle. Bacillus subtilis is a non-pathogenic bacterium found ubiquitously in the environment. However, genomic DNA sequencing of the specific isolate of Bacillus subtilis discovered within the Post and Brokaw powders reveals that it is genetically distinct from other known isolates of Bacillus subtilis. Analysis of the Bacillus subtilis from the Post and Brokaw envelopes revealed that these two isolates are identical.

In contrast to the highly detailed description of the genetic analyses carried out to pinpoint a set of four mutations present in the population of B. anthracis spores in the RMR-1029 flask and the subsequent comparison of this profile to other anthrax cultures in order to confine this particular profile only to cultures known to be derived from RMR-1029, the Investigative Summary has only one passing reference to B. subtilis (page 18, from list of suspects investigated and cleared):

A foreign-born scientist with particular expertise working with a Bacillus anthracis simulant known as Bacillus subtilis, and against whom there were allegations that s/he had connections with several individuals affiliated with the al-Qaeda and Ansar al-Islam terrorist networks.

Ah, but notice what has changed here in the description of B. subtilis. Instead of being "ubiquitous" and a "contaminant", this passage admits that B. subtilis is often used as a laboratory simulant of B. anthracis.

Clearly, if the FBI could have identified the strain of B. subtilis detected in the attack letters as available to Ivins, that finding would have played a prominent role in the Investigative Summary.

Even though Ivins can’t be linked to the particular B. subtilis strain used, there is a documented case of B. subtilis being used as a B. anthracis simulant at another facility where we already know that much of the material that went into RMR-1029 was produced. Recall from this diary that I analyzed the available information about the amount of B. anthracis used in the attacks and found it highly unlikely that Ivins could have cultured the large amount of spores used in the attacks with the equipment and time he had available. Much of the material in RMR-1029 was produced at Dugway.

On December 13, 2001, Judith Miller published an article in the New York Times, where she disclosed that "government officials have acknowledged that Army scientists in recent years have made anthrax in a powdered form that could be used as a weapon." She further pointed out that this work occurred at Dugway in 1998. It should be noted that the anthrax produced at Dugway for Ivins that went into RMR-1029 was cultured in 1997.

The Miller article then goes on to quote scientist William C. Patrick on how he coached scientists at Dugway in drying a pound of highly purified anthrax spores in 1998. Miller quotes a Dugway spokesperson as saying a strain different from Ames (the parent of RMR-1029) was used in the drying experiments. Note that a pound of spores is enough dried spores to produce hundreds of letters with the one to two grams of dried spores thought to be in each letter. But in a further bit from the Dugway spokesperson, we have this:

She said Dugway did make one- pound quantities of Bacillus subtilis, a benign germ sometimes used to simulate anthrax.

We know from scientific results discussed in this article that the material dried in 1998 is unlikely to have been in the letters because radiocarbon analysis suggested the attack material was no more than two years old in late 2001. However, both the facilities at Dugway and Batelle appear to have been carrying out anthrax projects in the appropriate time frame. Exactly one week prior to the 9/11 attacks, Judith Miller published two articles on biowarfare. The longer article discusses how US germ warfare research has pushed the limits of international law. She first describes work attributed to the CIA, but near the end of the article we have this, regarding work to replicate a feared Russian engineered strain of anthrax:

Eventually the C.I.A. drew up plans to replicate the strain, but intelligence officials said the agency hesitated because there was no specific report that an adversary was attempting to turn the superbug into a weapon.

This year, officials said, the project was taken over by the Pentagon’s intelligence arm, the Defense Intelligence Agency. Pentagon lawyers reviewed the proposal and said it complied with the treaty. Officials said the research would be part of Project Jefferson, yet another government effort to track the dangers posed by germ weapons.

A spokesman for Defense Intelligence, Lt. Cmdr. James Brooks, declined comment. Asked about the precautions at Battelle, which is to create the enhanced anthrax, Commander Brooks said security was ”entirely suitable for all work already conducted and planned for Project Jefferson.”

The second, shorter article discloses construction of a model germ warfare production facility at Dugway:

In a nondescript mustard-colored building that was once a military recreation hall and barbershop, the Pentagon has built a germ factory that could make enough lethal microbes to wipe out entire cities.

Adjacent to the pool tables, the shuffleboard and the bar stands a gleaming stainless steel cylinder, the 50-liter (53-quart) fermenter in which germs can be cultivated.

The apparatus, which includes a latticework of pipes and other equipment, was made entirely with commercially available components bought from hardware stores and other suppliers for about $1 million — a pittance for a weapon that could deliver death on such a large scale.

Miller goes on to claim that anthrax was never produced at the facility. Note that in my previous diary, I calculated that about 72 L of fermentation capacity would have been needed to produce the spores used in the attacks, so that would be just two "runs" of a fermenter the size of the one in this facility.

A key quote relating to the facility says "The project also showed us how relatively simple it would be for a terrorist to assemble such a facility without being detected". That’s very interesting given this bit from the Chemical and Engineering News article linked above:

Both Meselson and the former military scientist agree that making the purified preparations didn’t require an expensive laboratory setup. As the military scientist says, "A simple facility" is really all that’s needed. "I have concluded that maybe the hardest part is doing it safely so you don’t hurt yourself. Some experience is needed, but it’s probably more an art than a science," he says.

So, what we have now is the Defense Department taking over the lead on multiple anthrax projects from the CIA once the Bush-Cheney administration came into power. They inherited a demonstration facility at Dugway and they expanded a project at Batelle. And our primary source for much of this information is Judith Miller, who is infamous for her work in disseminating Defense Department lies about weapons of mass destruction in the lead-up to the invasion of Iraq.

Given what we know about the scientific analysis of the spores used in the attacks, culturing the spores is much more likely to have occurred at Dugway or Batelle than in Ivins’ lab. If we are to believe the isotopic analysis carried out (see the C&E News article) then Batelle seems more likely, but as I have previously pointed out, pre-packaged sterile water and culture media are readily available commercially from sources in the northeast,and would have produced the result seen even if used at Dugway. In fact, if the "simple" facility at Dugway were used, it seems likely that pre-packaged water and media would be employed. We also know that Dugway did extensive work with B. subtilis. The search warrant affidavit does not go into detail regarding the statement that the B. subtilis recovered from the attack spores did not match known isolates. It seems unlikely to me that a clandestine Defense Department program that was pushing the limits on legality would be entirely forthcoming in sharing its entire culture collection with an FBI investigation.

In conclusion, I fail to see how the FBI has eliminated the possibility that the spores used in the attacks were cultured and dried at Dugway or Batelle, with the Dugway "demonstration" facility being the most likely because of its remote location and secret status. Further, the disappearance of discussion of B. subtilis in the Investigative Summary is highly suspicious, and could reflect uncertainty on the part of the FBI that an exhaustive analysis was carried out to identify potential sources.

Under such a scenario, two batches of anthrax spores, using RMR-1029 material (known to have been shared with Dugway) to inoculate (start) the cultures, could have been grown in the demonstration facility, cleaned thoroughly, and dried. The spores for the Post and Brokaw letters would then have been mixed with a crude harvest of B. subtilis, used an "extender" or "carrier" to conserve the supply of purified material, which was used in its pure form in the Daschle and Leahy letters.

Alternatively, the two fermenter batches could have been harvested and purified separately, with the first batch having B. subtilis present either from improper cleaning of equipment after test runs with it or its inclusion as a carrier. The second harvest would then have produced the highly purified material in the second wave of letters.

We may never know what really happened. Given the history of deceit on the part of the CIA, the Defense Department and Judith Miller on the issue of weapons of mass destruction in general, discerning what is true in the various leaked reports on anthrax culturing and processing technology at the facilities at Dugway and Batelle will be difficult without Congressional hearings conducted under oath and threat of prosecution for perjury. Nevertheless, I remain unconvinced of the guilt of Bruce Ivins and strongly suspect a [non-USAMRIID] Defense Department, or a [non-USAMRIID] Defense Department-contracted source for the attack material.

Oh, and adding to the misdirection here, don’t forget the nonlethal fake anthrax letter that Miller received on October 12, 2001. I wonder how much analysis was carried out on that letter. There is nothing in the Investigative Report about it, either.