Wednesday, September 24, 2008

Additional comments by Dr. Popov on producing anthrax

1. I agree with all scientific conclusions [of the Analytical Chemistry article] except for the one that the silicon in the spore coat excludes its artificial origin. Sandia people think about the exosporium as an absolute barrier for small molecules but it is a diffuse, loosely-bound, and permeable layer. We can think about the spores as impregnated with the silicon compound. It may be true that the silicon did not help make the spores more dispersable, but it was added on purpose. See the following:

PERMEABILITY OF BACTERIAL SPORES II. Molecular Variables Affecting Solute Permeation. Philipp Gerhardt and S. H. Black, Department of Bacteriology, The University of Michigan Medical School, Ann Arbor, Michigan. J Bacteriol. 1961 November; 82(5): 750-760.

2. In order to test my scenario of the inconspicuous preparation of anthrax powders in the lab by somebody like Bruce Ivins, I estimated the amount of spores required for one letter. The information on internet says that the letters contained about seven to ten grams of material, of which roughly two to three grams were weaponized spores . Federal investigators say the Leahy anthrax powder had not been lost in the letter's opening. The amount, typical of the tainted letters, was 0.871 g .

So, let’s assume it was 0.9 g. According to Dugway and my estimates of the spore weight (based on the spore size of 1 micron^3) one gram of dry spores contains from (0.7 to 1)x10^12 spores. It is (0.6 to 0.9)x10^12 spores/letter. If the Bruce anthrax was 3x10^9/ml, it would take him at least 200 ml of the spore suspension per letter from the flask he possessed. For all five letters, he must have used up the whole one-liter flask. The solid medium process in the lab gives us 5x10^9 spores from a regular Petri dish. It would require at least 100 plates/letter. This number of plates is impossible to handle inconspicuously. In any case, if the amount of powder in the letter is correct, and the spores constitute the majority of it, there is more than a 10-fold discrepancy between the required amount of spores and the amount the perpetrator could have covertly taken from the flask or prepared on the agar plates. This bolsters a hypothesis of the fermentor-cultivated spores at the microbiological facility.

Interestingly, the powder from the first letters sent on Sep 18th to NY contained a lot of unsporulated bacteria. It is impossible to imagine that the powder of this quality could have been prepared for the military experiments by knowledgeable personnel. They would certainly discard the prep. Was the perp in rush to prepare the spores as quick as possible to make a connection with 9/11and therefore, by mistake, stopped the fermentation before the culture sporulated completely? It was only a week between the bombing and the mailings – very tight but possible schedule for this kind of job, if all the equipment was readily available. The next preps were more successful, but took longer. Has the equipment been previously used to cultivate B. subtilis for training purposes? This would be consistent with a contamination. Again, it indicates availability of a facility, and I’m afraid to say – a team effort, which is something fundamentally different from a lonely Bruce using Petri dishes and alyophilyzer.

3. The perpetrator did not have to use plates, but it is the simplest way. However, I disagree with the investigators’ time estimate. 3 to 7 days for several grams of spores? Have they tried to do it themselves? Running a fermentor and drying spores is not a 3-day job. It is not enough time even for a growth and sporulation on plates (we harvest spores on the 5th day). As I said earlier, a week is a very tight schedule. For a fermentor, there are additional steps of growing the seeding cultures (one or more days, depending on the volume). And fermentation cannot be accomplished during the evening hours only. By the way, did Bruce have access to the fermentor?

The theory of fermentor can only stand if other people were aware of the perpetrator’s experiments. If we accept this, we ought to conclude it was a collective effort at the well-equipped facility: it wasn’t just Bruce alone.


Anonymous said...

How about if we just follow the lead of the Judiciary Committees who questioned FBI Director Mueller last week about Amerithrax? The questions pertaining to the validity of the investigation were few in number and sounded one theme, best expressed in Congressman Nadler’s query: How did the FBI exclude the involvement of Battelle and Dugway? Mr. Mueller’s oft-repeated reply: I will have to get back to you on that.

Senator Leahy was the most pressing – after all, he was the addressee of one of the letters. His query: Where else besides Battelle and Dugway is anthrax weaponized? Chairman Leahy rejected Mueller’s “get back to you on that.” The Senator told the FBI Director to come back after the break with an answer. Lo and behold, the FBI director comes back with an answer: As to where else anthrax is weaponized, the answer is classified.

Oh my.

I am not a trained scientist, though I am quite capable of digesting technical matter. By profession, I am a lawyer practicing in Frederick, MD (home of Fort Detrick and the late Bruce Ivins). I am also a saxophone player and an organic gardener and a life-long peace activist who believes that we are running out of time within which to insist upon and act upon the truth. The issue of exactly what were the nature, extent, and sophistication of the processing that went into the anthrax in the Senators’ letters – that issue is getting more attention than it deserves. We know that (at least) nine people contracted inhalational anthrax, and that five people died from it. We know the anthrax was substantially processed, that it was remarkably pure, and that its strain and the presence of silicon aren’t uniquely but are distinctly American.

In fact, the issue of whether the anthrax letters originated in the U.S. of A. has been officially determined, presumably based on all of the information available, including the classified information. This determination was publicly made known very soon after the anthrax letters – that this was an “inside job.” In 2002, there was some blatant disinformation disseminated by media and government about the anthrax letters (ex. ABC News and bentonite), which disinformation played an obvious role in an across-the-board fraudulent sale of the Iraq War. But the years-long case against Hatfill relied upon the domestic determination, as does the officially dispositive case against Ivins.

The reason it took seven years for the FBI to (almost) close this case is that practically at the start of the investigation, the most likely source of the anthrax was declared off-limits – it was “off-limits” because it was classified. The “most likely source” is the place where for at least four years just before the anthrax letters, anthrax was being weaponized in classified programs. Senator Leahy actually referred (without explication) to the Sept. 4, 2001 article by William Broad at, which article is all about these up-to-then secret weaponization programs. All of these programs, whether sponsored by the CIA in West Jefferson, Ohio, or the DIA or the Army at Dugway, all of the laboratories involved, were ( and probably still are) operated by Battelle.

Furthermore, the FBI knows perfectly well from Dr. Ivins’ logs that Bruce Ivins (as ordered) was sending the celebrated RMR-1029 to Battelle periodically from 1997 into the summer of 2001. Nevertheless, the FBI wants us to share in its excitement about its “new branch of science – microbial forensics,” though it knows full well that all that said science does for us in the anthrax investigation is to narrow the number of suspects down to somewhere in the hundreds.


P.S. The news article from the journal Analytical Chemistry posted Sept. 23 by Meryl repeatedly quotes “James Burans of the National Bioforensic Analysis Center (NBFAC).” This is the same expert James Burans who told us as a part of the FBI science briefing on August 18 that the silicon in the anthrax letters was a “natural occurrence.” Anonymous in the first comment to the Sept. 23 posting refers to the FBI’s “witness who observed the Leahy letter being opened and says ‘It just had the consistency of a fine powder—nothing unique or distinguishing,’” -- said FBI witness is none other than James Burans. James Burans is indeed affiliated with the National Bioforensic Analysis Center (NBFAC). But the management and operation of the NBFAC is contracted out by the Department of Homeland Security to Battelle, and James Burans is a Battelle employee.

Anonymous said...

We don't know when the anthrax was made. It's wrong to leap to the conclusion that it was made after 9-11. The envelopes were filled after 9-11, but the anthrax could have been made long (up to 2 years) before.

Ellen Byrne said...

Barry - an excellent call to arms!

Tis is worse that Watergate. Bigger crimes, bigger cover-up.

daedalus2u said...

My understanding of why liquid fermenters are used to grow anthrax for bioweapons is for scale-up. Liquids are easy to pump, there is lots of scale-up information on liquid fermenters, there is lots of experience with liquid fermenters. That is how beer and wine is produced, how yogurt is produced, how many pharmaceutical intermediates are produced including bulk commodities such as citric acid and vitamin C.

The concentration of bacteria and hence of spores in a liquid fermenter is limited by the concentration of substrate that can be achieved. The substrates for anthrax growth are amino acids, carbohydrates, lipids, minerals and such. That substrate concentration is limited to perhaps 10 grams per liter (to avoid precipitation and clogging of equipment, something you really don’t want to have happen in your bioweapons factory). The rotor router man charges more for unclogging bioweapons factories.

The normal environmental niche for replicating anthrax is inside mammals. Inside a mammal, the substrate concentration is virtually 100%. Anthrax is anaerobic. It is exposure to oxygen that causes sporulation.

If instead of using a liquid media you used a solid or semi-solid media, what concentration of anthrax bacteria and anthrax spores could you reach? What is it that would limit it? If you optimize the nutrient media, such that there is no single nutrient is limiting, that concentration could be very high. In raising animals for food, a number that is important is feed conversion ratio, or the ratio of how much live body weight is produced per unit weight of feed. For farmed salmon this ration is about 1.2, that is 1.2 kg feed produces 1 kg live weight. Bacteria are a lot simpler than salmon and their conversion of utilizable substrate into biomass is higher. It should be pretty easy to get 10 grams of spores from 20 grams of dry media.

Petri dishes are used for isolating and identifying bacteria. Growing bacteria on the surface of the solid media makes sense because they are easier to then identify and isolate as a pure culture.

Old Atlantic Lighthouse said...

These are very good comments by Dr. Popov. They basically show that the FBI thought hours late in lab equals could have made the anthrax in the letters.

The paper I referenced earlier

Production of Bacillus Spores as a Simulant for Biological Warfare Agents
Authors: Laurie F. Carey; Diane C. St. Amant; Mark A. Guelta;

now appears to be here for 15 dollars as a pdf (I haven't downloaded it)

It now appears to be gone from its free down load page.

Looks like the powers that be don't want the people to read this paper.

This paper came out in 2004. Was the idea then that it proved someone in a lab like Detrick could not have produced the anthrax because the amounts of plates or liters of CD would be too large to escape detection by coworkers? Did the FBI intend to introduce that paper into testimony in a trial against Hatfill to eliminate all the Bruce Ivins the defense would want to say did it? Now they are hiding the paper?

Old Atlantic Lighthouse said...

Biosafety level 3 and 4 do allow liquid nitrogen but there are still precautions required.

Search biosafety level "liquid nitrogen"

"Handling frozen cell cultures

Storage and retrieval of frozen cell cultures from liquid nitrogen requires appropriate Personal Protection Equipment (PPE). The three major risks associated with liquid nitrogen (-196� C) are: frostbite, asphyxiation, and exposure. Gloves thick enough to act as insulation but flexible enough to allow manipulation of ampoules should be worn. When liquid nitrogen boils off during routine use of the freezer, regular ventilation is sufficient to remove excess nitrogen, but when nitrogen is being dispensed, or a lot of material is being inserted in the freezer, extra ventilation will be necessary.

When ampoules are submerged in nitrogen, a high-pressure difference results between the outside and the inside of the ampoule. If it is not perfectly sealed, this results in inspiration of liquid nitrogen which will cause the ampoule to explode violently when thawed. Wear eye protection and a face shield. This can be avoided by storing the ampoules in the gas phase or by ensuring that the ampoules are perfectly sealed. Thawing from storage under liquid nitrogen must always be performed in a container with a lid, such as a plastic bucket and eye protection and face shields must be worn.


Use of liquid nitrogen in a lyophilizer for drying anthrax "broth" would be noticeable to lab workers. They do have to maintain a sense of what is going on in the lab, and because liquid nitrogen is a hazard, they will tend to maintain awareness of it.

Ft. Detrick did say a lyophilizer had to be checked out. Did they allow it in Suite B3? Were there rules on it?