In-situ Concrete Repair via GMO Bacteria

it sound good. I think more appilcations could be found like in pulmbing and in under ground water tanks. I wonder about time needed to do repair. What is the feeding culture to grow this Bacillus species? How it can achieved uniformity of the crack repair? I think there might be some pores resulting from biological desintegration of protiens on the cell wall of the bacteria.

Thanks Rorschach..

Interesting. I just posted a response to another thread submitted the same day as your submission about a leaking reservoir. I had recommended he contact the school but also gave an alternative. I was not sure of the potable water issues involved but once the bacteria have left, I see no conflict. Funny, it takes oil well or water well workers to take serious interest in the microbiology of their field. Ground water microbiology can mobilize many metals and often treatment of the metals ignores the root cause. The bacteria are the real rulers of the earth and their use to repair concrete is no surprise. Good digging Rorscharch, keep up the interest.

I took a little time to read about B.subtilis and refresh myself on the genetic modification process (not my area of expertise). There's another species in that genus that grows at cement pH so I guess that's where they got the control gene.

I see that Bacillus subtilis is a model organism in the lab, partly because it is not pathogenic, partly because it is "competent" for integration of foreign DNA naturally during its life cycle, therefore easy to modify, and partly because it has the propensity to form biofilms - one of the properties involved in the cement patch formation. There has been knowledge of and interest in the biofilm forming properties for engineering purposes for more than fifty years, but it was not exploited in the past because of hemolytic (blood cell breaking) properties associated with the same genes. The hemolytic effects occur when the same genes are combined or expressed in specific ways - this is also known from pathogenic species in the same genus (eg anthrax).

I also see that the wild Bacillus subtilis strains are found as highly beneficial symbionts in a lot of places: as an endophyte in Mulberry trees preventing wilt, in the soil where it has other anti-pathogen agricultural applications (damping off, fusarium), in animals eg shrimp where they protect against Vibrio infections, and in the human gut and on human skin, protecting against fungal infection. B. subtilis is even a food supplement.

The cement patch is a very elegant bioengineering design, even using the research on quorum sensing to engineer the patch. I am amazed it has the strength of concrete - quite a feat!

My only question would be, if the genome of B. subtilis is so readily modified, ie without intervention, occuring naturally in its life cycle, then is there any reason to be reassured it would not under any circumstance drop the pH control gene and end up mixing in the wild pool which is in the environment everywhere, carrying a gene design that promotes formation of a cement-like product?

I wouldn't want cement forming in my gut, nor on my tomato roots or inside my mulberry trees. What assurance do we have that this can't happen?

Therein lies my concern. I'm not confident that the pH control gene is sufficient to prevent the bacteria from plasmid swaping with wild strains and result in a new species that may not be easily stopped since biofilms are notoriously difficult to sterilize/control. Cement forming in arable soil would be a problem. cement forming in your gut may or may not be a problem (depending on how quickly if forms, and whether gastric fluids will attack the cement or not.)

And from a purely practical point, what about dirt and debris that gets down in the crack? Wouldn't that create voids/delaminations that would weaken the reapair?

I am less concerned about wild GM bacillus from the cement curing entering the human GIT and creating problems. These GM bacteria are not able to withstand the low pH found within the GIT. The pH of cement curing is very alkaline whereas the pH of the stomach is very Acidic. They are polar opposites.

From the report:

"they have a built-in self-destruct gene that prevents them from proliferating away from the concrete target."

Another saving factor is the presence of peroxidase and catalase enzymes within the bacillus bacteria. These enzymes are also present in every cell within the human body. Hydrogen peroxide is broken down by these enzymes. As the bacillus bacteria die during the cementing, they discharge peroxide. To protect each abutting cell from damage the enzymes on the adjacent cell destroy the peroxide but as more and more of the bacillus die (they are essentially starved and dehydrated), there is more peroxide to the point of overwhelming the cell and thus the peroxide can now lyse the cell wall of the unprotected cell. This lysis with hydrogen peroxide is the mechanism within the human body to get rid of unwanted cells.

The consortium of bacteria found within most biofilms contain bacteria with and without the peroxidase or catalase enzymes. All aerobic cells will contain the enzymes while none of the anaerobic bacteria will contain the enzymes. There are a host of faculative bacteria found between the aerobic and anerobic microorganism. These may or may not have enzymes but are capable of aerobic or anaerobic conditions. In ground water environments, where biofilms and biofouling (excessive biofilm) are common, the use of hydrogen peroxide at a dosage of about 800 mg/L will break down most of the biofilms for the same reason stated. The aerobic and faculative bacteria are overwhelmed whereas the anaerobic bacteria have no enzymes (obligate anaerobe) and any oxygen is toxic to them. The addition of peroxide to a well on some sort of regular bases is a prudent practice that will control biofilms that can lead to biofouling. Biofouling ( biofilms that are out of control and now interfere with quality and quantity of water) may require additional mechanical treatment and chemical treatment.

I see no reason that the pH-tolerance gene should not be swapped out like any other. It is already one of the genes swapped out to make the cement-patching GMO, so exchange of the gene is clearly possible. Your comment about dirt suggests a case in point.

While I don't know whether unavoidable amounts of dirt would be enough to weaken the repair, mere traces of any soil whatsoever would virtually guarantee that a wild type Bacillus subtilis is present in the crack to provide the alternate pH-tolerance gene for a swap. That is because this organism is so widespread in soil, its presence is virtually assured pretty much anywhere afaict.

The wild type would not do well in concrete per se, but a crumb of soil is plenty of real estate on the bacterial scale. It would only take some incidental water during the patch to wash out a transformed GMO-wild type into the environment.

It's reported that the wild type is generally less "competent" to accept foreign DNA than the lab strains, which are very easy to modify. So it's more likely that wild DNA would be picked up by the GMO variant by horizontal transfer - a dead B. subtilis would be as serviceable as a living one to provide the alternate pH gene, so simply sterilizing trace dirt in the cracks wouldn't prevent it either.

"a dead B. subtilis would be as serviceable as a living one to provide the alternate pH gene, so simply sterilizing trace dirt in the cracks wouldn't prevent it either"

I do understand the resilience of bacillus type of bacteria in the environment. They have evolved to withstand a lot of different environments such as excess UV exposure. The cell walls are very hard and somewhat along the lines of a walnut shell. That is part of the reason they are so prevalent in soils. They can be harboured inside the outer shell or wall for centuries (anthrax). It is also used as a target microorganism for disinfection (B. histolyica). In a repair of cement environment, the mobility of the microbe would be most difficult. The Bacillus microbe is not necessary viable after its use in cementing. I am not familiar enough with the process from the Scots but I would be more upbeat and less concerned about releasing/creating cementing microbes in the environment. Darwinism is a constant in the environment and no doubt conditions surrounding any organism will promote evolution in a different direction. This is happening all the time. In a natural system sterilization of dirt in a cracked cement structure would be impossible or at least very hard to accomplish and verify. It is very likely that the wild B. subtilis will consume the cementing ones. I say this without proof but the wild bacteria have a good track record.

Even if they did become prevalent, they would not cause human grief but may cause some harder clumped soils locally, I think. An interesting story on the discovery of B. subtilis can be read here. I am not sure who first tried this cure, but they had to have been brave. I will try to remember it when I am suffering in the desert.

Art, you are providing intelligent and fruitful comment. I do not discount your concerns, but I have my doubts of GM bacillus becoming a problem. Human initiated Genetic modification has been with us for some time. So we are now the big experiment in using GMOs. The modified B. subtilus is just another cog in a movement that will likely never stopped. At least we are not consuming it.

Kevin, I do agree with you that it's a pretty amazing engineering feat. I like to base my assurance of safety on factual assessments and answers - I just didn't have those answers so I speculated.

Hazard spotting is something I consider a worthy sport - a mental exercise that develops into a skill the more it's practiced. In this case, the concerns I raised were largely due to ignorance of the details of the process, which I cleared up by searching for the original authors of the work.

The "cement" equivalent produced in the patch is just calcium carbonate and the polysaccharide levan, so there is no reason to anticipate any health or environmental hazard from the escape of a cement producing gene cluster into the wild, if this did or could occur.

There are multiple layers of protection against escape into the wild: dependance on tryptophan for growth: dependence on sucrose for levan production: and kill switch activation by depletion of sucrose. The crack filling process is clearly limited by the tryptophan and sucrose provided in the inoculation package.

On that basis I've concluded that this seems like a very safe application after all - kudos to the designers!