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.