Friday, September 19, 2008

Comments by Professor Sergei Popov on anthrax and on Dr. B.H. Rosenberg's paper

Dr. Popov worked in the former Soviet Union's Biopreparat Program and is a professor at George Mason University. Having met with him several years ago, I can attest to his impressive knowledge of anthrax. Here he demonstrates a deep understanding of the principles of weaponization.

Some of his other comments include the following:
1. The Sandia pictures completely agree with my expectation of partially collapsed exosporium. It may be still there but hard to detect in the dry spores. Treatment of the B. subtilis spores with urea helped reveal the exosporium-like layer (A picture from Nature 263, 53 - 54 is on the second of the attached slides). Please notice the similarities with Sandia spores. This technique could be useful to determine the amount of exosporium in Sandia samples. Your calculations present a strong argument that the silicon content is unnaturally high. However, I’m in doubt that the highly variable amount of Si in the samples can be used as a reliable forensic marker on par with genetic ones. The anthrax attack was a deliberate action, and there is nothing surprising to learn that the spores were treated deliberately. Can this conclusion help identify a particular perpetrator?

2. The nature of debris clearly visible in the spore prep is intriguing. It looks quite similar to dry agar from a Petri dish

3. I think you may overestimate the technical difficulty of the siliconization. It may be just a drop of an old-style school glue (liquid glass). A well-developed technology is supposed to give consistent results, but we see a high variability of the Si content, which indicates experimentation with different treatments. In your opinion the perpetrator is primitive and this effectively exculpates Detrick. In my opinion, even at Detrick there could be people with a creative mind who know chemistry beyond written protocols.

4. I don’t know what the FBI has tried for reverse engineering. Did they even consider soluble silicates, not siloxanes? I guess we have to admit that the perp was/is a clever guy. Why should he leave a trace ordering a reagent if it was available from Wal Mart? The person obviously realized the possibility to be traced back and therefore took advantage of his unique knowledge of how to accomplish his task in the most inconspicuous way possible?

5. As I wrote, a familiar example is the drug Simethicone, which is the active ingredient in drugs such as Gas-X. Simethicone is generally available over the counter under many trade names in varying dosage sizes, including: Flatulex, Baby's Own Infant Drops, Gas Relief, Gas-X, Genasyme, Maalox Anti-Gas, Maalox Max, etc. The use of antifoaming agent such as Simethicone will result in detectable Si without other inorganic component. I’m not talking about a paper glue, which is a pure sodium silicate.

15 comments:

Any of N said...

Popov's final set of comments in the linked document are quite intriguing, especially the last paragraph. Could it really be that easy to produce anthrax powder like the batches sent in the mail? I'm terribly naive when it comes to the science in this area, but the scenario Popov outlines sounds like it may be plausible.

We will have a very mixed bag of implications if Popov's guesswork is on the money. On the upside, we can pretty much stop worrying that a state sponsored effort is responsible. (And wondering which state it might be.) On the downside, we can start distrusting our news sources even more, because the difficulty in making the anthrax powder has always been presented as if it were one of the tenets of the case. And wouldn't it also be true that somebody else could easily whip up a batch of deadly anthrax powder? A few spores to start with, some agar plates, an empty vial, a plastic container with desiccant, a drop of silicate, and voila!

Ivin's guilt would be a little easier to believe, but it would also mean that many other people could have pulled it off. Or am I way off base with all this?

Ellen Byrne said...

This is all so above my head. The American people deserve an INDEPENDENT panel of EXPERT scientists and detectives to examine EVERYTHING concerning this case.

It took Steve Hatfill all these years, a fighting spirit and hundreds of thousands ot dollars to clear his name though he will be forever linked to the mailings.

Americans deserve to know if the powder was naturally occuring and if we should all be on Cipro or a vaccine.

daedalus2u said...

I don't think it is correct to characterize what Popov has to say as "guesswork". He strikes me as someone who knows the ins and outs of anthrax growth, sporulation, and conversion of those spores into a weapon.

It has only been those who are not knowledgeable regarding anthrax weaponization who have said how difficult it is. If you don't know how to do something it is always difficult. Once you do know how it becomes trivial.

Most of the difficulty in replicating something (maybe 90%) is simply knowing that it was done in the first place. The idea that there are arcane "secrets" on how to make WMD is nonsense put out by politicians and other non-experts.

Why didn’t they trace the genetics of the B. subtilus? If, as was stated, the strain was “unique”, that would provide enormous specificity in confirming the source.

Anthrax is a pathogen of mammalian tissues. Normally those tissues have very low silicon content. For silicon to be incorporated into the spores “naturally”, what does that mean? That silicon was incorporated into the anthrax as part of its normal physiology? Are pathways for silicon metabolism in anthrax known? Does anthrax actually incorporate silicon? Some organisms do incorporate silicon, such as diatoms. Silicon in organisms is only found coordinated to oxygen, never to carbon or hydrogen. What silicon is coordinated to conceivably could be measured using the fine structure of the x-ray emission spectrum. That takes a significant amount of material (maybe a gram), but is in principle non-destructive. That should be able to distinguish between the Si-(O)4 of silicates and the Si-(R)2(O)2 of siloxanes and the Si-(H)2(R)2 of silanes. Similarly a mass spec analysis would show if any silanes or siloxanes were present.

The outside of bacteria is a somewhat diffuse barrier of lipopolysaccharide; a combination of lipid (which is hydrophobic) and sugar chains (which are hydrophilic). This is where a silicone or silane would tend to collect.

Old Atlantic Lighthouse said...

This is a very good write up. On the silicon. There are several possibilities to consider. Note all of the following apply whether the silicon is strictly inside, strictly outside, or some of both, possibly dynamically at different stages of growth, preparation, storage, and deployment.

1a. It was accidental. 1b. Or it was a guess first time out.
2. It was based on know-how from some source to the preparer.
3. It was based on trial and error by the preparer using a lab setup similar to that in the paper at the end. This could use anthrax substitutes to test. This might take a long time.
4. It can still be done with simple methods, but the know-how has to be developed with a combined team of science including physics know-how and equipment.
5. It requires elaborate equipment to prepare.
6. There is intense physics know-how and equipment in the development and preparation.

If its easy to try out table top methods with anthrax substitutes at home using petri dishes and one of them works and uses ready available materials that don't leave a trail, then that tends towards Ivins being possible. That also lets open the bag for several others as well.

Another scenario is that a government spy agency told its weapons labs to find a recipe for agents in the field to use quickly from available resources. I.e. the spy agency gave a team of scientists the job of coming up with a recipe like the one in the paper at the end. That might have required physics know-how and equipment or might not, just a lot of trial and error.

Old Atlantic Lighthouse said...

Why subtilis? Consider the agent scenario. A field agent as part of the procedure buys equipment and materials and then does a run with
subtilis to check its working. Then they do their run with anthrax. Of course, amateurs might do this for the same reasons depending on their situation.

Old Atlantic Lighthouse said...

"Take a small amount of spores and inoculate several agar plates (this is a part of a microbiological routine). Nobody will notice the “missing” amount of bacteria. Forget about the plates for several days in order to let the culture grow and sporulate. If somebody finds the plates and asks questions, say “sorry, I forgot to dispose of them” (it happens all the time). If not, scrape the spores, which are now almost “theoretically” pure, and put a paste into an unsuspicious container. It may be an empty vial left after a used reagent. Plenty are lying around. Attach a cap loosely and put a vial into a bigger container
with some desiccant in it. These tin or plastic containers come with many reagents and protect the content from moisture. Close the lid and put the container aside, and it will look like somebody forgot to dispose of it. Again, if somebody asks, say it is trash. Two days later, stay late in the lab, take the vial out, use a spatula to disperse the dry stuff, and put it into an envelope. Next time, you may try to add a drop of silicate to the spore paste before drying. All these procedures will take minutes to accomplish. Everything is disposable, and there will be no traces left behind. Now, try to criticize this scenario, and maybe we will get a little bit closer to what really had taken place."

The cycle time takes 1 week let's say assuming he typically takes weekends off.

This is one recipe. So one recipe takes one week to test. He can add something during growth, i.e. on the Agar or in the drying tube. So there are two points for addition. (He might also add something during those days, which can give us up to 4 or 5 insertion points, which can be of different amounts and different compounds.)

Suppose we want to use only what is in a drugstore. We might have 50 things to try for each insertion point and a varying amount of each. So with 2 points and one drop only at each, we have 2500 combinations. Each one takes a week to test, so that is 2500 weeks.

He can't save the results, label them and store them to compare different combinations. He can't produce an aerosol in the lab each week or people will start to notice powder sticking all over the place. Also the trash excuse stops working after people notice this week after week. So how does he test the different combinations? He has to destroy each trial each week and just remember without writing anything down? What if he has to make improvements in one batch that works well and combine it with know-how from another batch? He will need to keep records and really keep the powder in containers with labels how it was prepared.

So to find the recipe that works, he has to do a lot of trial and error. To cut that down he needs to use methods like imaging. If he can use physics equipment to image the powders he can see what is going wrong. If he has a chemist to help him he can get insights into the compounds. If he is worried about charges and diffusion he needs a theoretical physicist and an experimentalist.

So we are back to a multi-year project with a team of people and a budget and physics equipment.

Its clear that the public science of the US does not have the right recipe published. It seems likely the US wmd community doesn't know it. But Russia had many projects to understand this.

Interview of Dr. Popov on PBS.

http://www.pbs.org/wgbh/nova/bioterror/biow_popov.html

Dr. Popov points out he didn't know more than a tiny bit of what was going on in Russia, and that stopped when he left c. 1992.

"Popov: Well, I didn't know the whole picture. I didn't know the scope of research, but I had some indirect knowledge. And I realized that it was a huge, huge program which involved dozens of different institutions and facilities.

At my level, I knew what was the true purpose of the research, but sometimes I didn't know the final results of my work. "

The program that would have done the trial and error tests of what combination of drug store products to use to aerosol anthrax is the bio program of Russia.

Its clear at this point that the FBI, Sandia, Dr. Popov, and many others don't know what was done. They don't know who, what, where, why, how, when, of how it was prepared or how the recipe was developed, what the silicon did for the fluid or aerosol properties, or how it was discovered. Its a mystery to the experts in the US. That means it was probably done in Russia.

My code for word verification is kgbihdpb, I take that as a sign.

daedalus2u said...

OAL, I think you are greatly overestimating the degree of difficulty of getting something that is "good enough" for someone who has a good understanding of the physics and biochemistry involved.

The FBI didn't try to reverse engineer it, or to see how easy or difficult it would be to make something similar. The presumption that it is difficult is only an assumption by those who don't know very much about it.

By all reports, the first batch that was sent was pretty crappy and the later batches were pretty good, indicating a very steep learning curve. Silicones are very widely used to prevent things from sticking. Essentially all of the release papers on things that are self-adhesive are made to be non-stick through an application of silicones. Anthrax spores evolved to disperse themselves.

The degree of difficulty in making anthrax to be usable as a weapon is not in making a few grams of an easily dispersible powder. It is in making hundreds of tons cheaply in a form that has a known and long shelf life and can be delivered via munitions which also have a long and known shelf life and which don't pose an inordinate risk to one's own troops or factory workers.

Making the spores is the easy part, the bacteria do that all by themselves. It is making the spores into a weapon that has military utility that is difficult. Making a weapon that has military utility is so difficult that most nations have abandoned trying. The "problem" isn't in making weapons that are deadly; it is in figuring out how to make something that has military utility out of something that is so deadly and indiscriminate. If you want a weapon that will kill civilians indiscriminately, a bioweapon would work. Killing civilians indiscriminately is not a "legitimate" military objective. Anyone in the military chain of command who orders or who follows such orders is guilty of war crimes and of crimes against humanity. The only people in the military or in the government who would want such weapons are those who don't mind being war criminals; those are people we don't want in the military or in the government, the place for people like that is in prison.

There are similar "formulas" for making bacterial spores for use as insecticides in the patent literature available for free download anywhere in the world. The only difference is the type of bacteria used. Use Bacillus thuringiensis and you have an insecticide for corn borer, use Bacillus anthracis and you have a deadly bioweapon. Unless you have a pretty sophisticated lab you can't tell them apart.

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1657067

The idea that the perpetrator could only be some top secret genius level mad scientist is nonsense. The anthrax was sent for a reason, that reason pretty obviously was not to kill people, but to scare them. Why? So they would do something they wouldn't do if they were not so scared. Why have there been no follow on attacks? Pretty obviously because the first attacks reached their objective. The first attack against the tabloid was followed up with attacks against other media, and those followed up by attacks against Democratic government officials. No more attacks, pretty obviously the objectives were reached. The attacks didn't stop because the FBI caught the perpetrator but because the perpetrator's objective was reached. What has happened that has satisfied the perpetrator?

Old Atlantic Lighthouse said...

reply to daedalus2u

I appreciate your comments, but lets try to get down to numbers to follow your hypothesis of easy.

Click on handle at to read pdf:
http://oai.dtic.mil/oai/oai?verb=getRecord&metadataPrefix=html&identifier=ADA426293

I searched on: growing time bacillus spores.

They have very interesting results. The tables are very interesting. Are their results as good as what was mailed? They didn't try any silicon compounds or drying agents that I could find.

Remember one view is that the first batch was not as good because it was exposed to moisture. Is humidity enough? The first batch was sent Sep 18 when its more humid. The second was sent after Oct 6 when its less.

Even with the above paper, which is pretty good, we still don't have the information to answer all our questions. If it took 10 trials at one week each that is 10 weeks. Note the results on page 20 of the pdf. They have to use 11 vials in one case to get 1 gram of powder.

Last line of 5th partition is only 4 days but it takes a lot of vials to get less than one gram.

If the first mailing was after 9/11 he needed to get it done in less than one week. So 4 days is good for that. But it takes 9 vials for .319 grams so that means you need say 30 vials for one gram. 5 envelopes so 150 vials.

I don't know how big a vial is but 150 vials hidden for 4 days in the lab sounds like that dog don't hunt.

The line above is 10 days with only 5 vials to get .981 grams. But he didn't have 10 days.

Are the results reported good enough? Or are they inadequate? What about the aerosol results?

Some of these results they say they grew it one way and got no spores. That doesn't sound like this is so reliable in those cases.

How come we still don't know the answers? Do we add the silicon additive in the growth stage or drying stage? Do spores take up silicon in the drying stage? Or in the growth stage? Didn't Ivins have to find that out?

Also in Google Scholar search with Bruce Ivins as author, none of his papers has the word equation in it or inequality.

Your search - equation author:Bruce author:E. author:Ivins - did not match any articles.

Results 1 - 33 of 33 for author:Bruce author:E. author:Ivins.

So out of 33 papers he never used the word equation. How did a person who never used the word equation plan out all this? We see from the paper I link to that there are lots of variables to manage. Number of days, number of vials, percents, etc.

He was doing all these production runs, and had to control their quality while keeping it all hidden. The guys who did the paper didn't have to hide their stuff at the end of each day. Wouldn't that reduce the efficiency a lot?

If you only get 2 to 3 hours in the unit each night and have to spend time getting stuff out of hiding and hiding it at the end, doesn't that cut down your production rate and quality results?

If he had 150 vials that he had to move in and out of some place to do stuff wouldn't the spores get all over the place?

It seems Ivins had a more difficult work environment for production runs of his spores than the guys in the paper. He had to have more vials than they did but had to hide it all.

They did all sorts of tests with lab equipment to check their results. Some of their runs gave zero spores. Didn't Ivins have to do all the runs they did to find out what worked? Did he have the NSM they talk about? What were his available options?

"nutrient sporulation medium"

Results 1 - 52 of 52 for "nutrient sporulation medium"

Doesn't sound like every lab has it.

Your search - "nutrient sporulation medium" detrick - did not match any documents.

Its not looking good for what is claimed for Ivins from this paper. What do you think?

Old Atlantic Lighthouse said...

I've worked up some numbers from the paper. I estimate he needed 90 square feet of NSM plates to produce 5g for the 5 envelopes at 1g each. If it was 2 grams, he needed 180 square feet.

Bottom page 13, 1 gram of dry spores
per liter (meaning liter as ref on page 12).

Page 12 2.9.1 for one liter used 15 to 18 large 100 x 150 mm plates. i.e. 4 by 6 inch plates or 24 sq inches. So 15 of them is
360 sq inches. 144 inches in a square foot. 18 of them is 432 which is 3 x 144 or 3 square feet. Lets use that.

At 5g we need 15 square feet. But hold on, that's when you use the longer growth period in days. We have only 4 days. So we get our efficiency factor of 6, so we need 90 square feet for 5g. 90 square feet of plates.

Efficiency factor estimate: Go to page 20 and compare NSM 4 day v 10 day lines. 4 days takes 9 vials to get .3195 grams. 10 days takes 5 vials to get .9817 grams. Lets call this 10 vials to 5 vials and 1/3 to 1 gram. So we get a factor of 6 efficiency by the longer period.

So 1 gram of powder required 3 square feet of plates for the long period. 5 grams means 15 square feet of plate. But to do it in 4 days requires 90 square feet of plate.

So where did he put this 90 square feet of plate?

Old Atlantic Lighthouse said...

I'm not sure I did that scale factor correctly. I may have misinterpreted the table and some pairs of NSM give different results.

Even at 15 square feet its a lot. Also if he messed one up by getting the envelope wet after he put in anthrax he would have to redo it.

Did he use 2g per envelope the first time? That would take 30 square feet.

Old Atlantic Lighthouse said...

We can look at the table on page 20 for the CD results. Line 7 is the best case, .5 g for 1 liter of CD. So to get 5 grams, he needs 10 liters. So he has to hide 10 liters in the lab. Think of a 1 liter water sports bottle. So he has to hide 10 of these in the lab.

That assumes every bottle is a winner. Some of the lines got less than .1 gram for a liter after 7 or more days. Those are losers. If he gets winners 1/4 of the time, he needs 40 bottles. Remember the lines of 7 or more days to get a lot are not real winners since he can't wait that long.

Even at 10 big sports drinks bottles in the lab, that seems a lot for no one to notice.

Old Atlantic Lighthouse said...

There isn't much citation of this paper on the Internet:

Results 1 - 6 of 6 for "Production of Bacillus Spores as a Simulant for Biological Warfare Agents"

Results 1 - 5 of 5 for Carey, Laurie F. ; St. Amant, Diane C. ; Guelta, Mark A..

See page 28 for histograms of the size of the spores. The median is about 1 micron.

Search microns anthrax 2001. One finds references to 1.5 to 3 microns as the range. Thus these production tests were not the same range.

If he was to do production runs, he had to scale up from smaller runs. But how easy would it be to extrapolate from tiny runs? Or
were there earlier papers to give him estimates? Did he have copies of such papers? Talk to anyone about it?

Anonymous said...

Old Atlantic Lighthouse,

It's obvious you haven't been paying attention to the FBI scientists.

What don't you understand about "Everything was an accident"?

The unusually large silicon spike was an unreproducible accident of nature. The deadly particle size of the anthrax powder was an accident was caused by US Postal machinery. Ivins, a skilled microbiologist and expert on the production of pure anthrax, accidentally produced contaminated anthrax in his own lab.

It was all an accident.

OK?

In fact, the FBI will soon reveal evidence that the anthrax attacker didn't mean to make anthrax, and didn't mean to mail it either. A lab mix-up. An outgoing mail mix-up. You know how those things go...

Old Atlantic Lighthouse said...

OK. However, if we combine this paper with the FBI affidavit on hours in the lab we get some interesting insights.

The FBI says that after Sep 11, 2001 his lab hours at night started Sep 15, Friday. (He also had August hours, but I put those aside for now.) If he started Friday and grew anthrax and then dried it in time to mail Monday, he had to go really fast.

He had to get 5 grams at least of output if we assume 1g per envelope (instead of 2g which it might have been). So he had to do at least 10 liters starting Friday. So he grew them in one day and then dried them the next night and put the envelopes together on Sunday and mailed it Monday. For that to be the scenario, he had to have a fermenter with a capacity of 10 liters and a lyophilizer with a capacity of 10 liters net plus whatever liquid nitrogen it needed, so maybe 20 liters total if that is the way they measure it. He had to ferment and dry in one batch each in such short time.

So these are a really big fermenter and lyophilizer by lab standards. Or he did it in August and hid it for a month. But he still likely needed a fermenter and lyophilizer.

Lyophilizers use liquid nitrogen and there are warnings about injury from this. So lab workers are going to want to notice if the lyophilizer has liquid nitrogen in it and be told that by anyone using it so they don't injure themselves.

What is the status of FBI statements on a lyophilizer and a fermenter in Suite B3? They are not saying much. They keep going back to the DNA in the flask.

The second nights end Friday Oct 5. This is a problem for the FBI theory. If he used a lyohpilizer these nights then he had to leave it overnight and then other workers were in the lab.

Old Atlantic Lighthouse said...

I saw Dr. Popov's comments on a new thread which are valuable, but I will post this here since it relates to the paper linked above and comments on it.

The paper said that 1 liter of CD produces .5 grams in the best case or one of the best ones for short time periods. However, in other cases, yield is much lower. The highest yield for CD is .861 grams for one liter. However, many are much less.

If the typical yield was .2 grams then it would take 5 liters to produce 1 gram of powder. If we go with 1g per envelope, then we need 5 liters to start to end up with one envelope. So by this, we would need 25 liters. To some extent this applies, roughly, even to longer than 3 days.

Note the table and paper as I understand the paper did not use a fermenter.

25 liters can be thought of as 25 somewhat large size water bottles. They won't look like water though as I understand it. So he can't have taken ordinary water bottles, put the CD in them and taken them to his office and have water bottles sit there with no one realizing he is growing anthrax.

Also this quantity requires a large scale lyophilizer. Those use liquid nitrogen. One sees ratings of them like 4.5 liters. I interpret or guess that to mean the liquid nitrogen plus whatever is put in it all together are limited by 4.5 liters. So if one is batching say 2 liters of CD one might use 2.5 liters of liquid nitrogen, so the actual capacity of CD is only 2 liters per run.

These take overnight or longer as I understand it from my Internet searches. Also one finds cautions about liquid nitrogen causing burns or even worse lab accidents. So people in a lab want to know if there is liquid nitrogen in the lyophilizer as a safety measure. It wouldn't be possible to batch all this through the lyophilizer on weekdays without the others knowing.

To repeat a point I made before, Oct 5 is a Friday and lyophilizing is part of the final work. So if he was drying in the October week ending in Oct 5 he had to use the lyophilizer during the day with liquid nitrogen in it that his coworkers would be more likely to notice.

So far there is no solid evidence from the FBI that Suite B3 had a fermenter or lyophilizer (as opposed to a speed-vac) in it. There is also no information that the lyophilizer was of a size to handle 25 liters or more needed for the first set of letters. That is true even if the processing was done in August instead of September.