Debunking myths on genetics and DNA

Sunday, May 5, 2013

Pumping fuel from bacteria

In my last post I discussed a bioengineered E. coli strain capable of producing an engine compatible biofuel. I hailed the finding as more efficient than ordinary biofuels because this technique has less environmental impact than biofuels from crops, for example, or cellulose, which instead use great amounts of water and forest land.

I did some more reading on the topic and found out that, surprise surprise, there are some costs in harvesting biofuels from bacteria as well, so my discussion was incomplete. However, there are good news at the horizon.

When I first read the Howard et al. paper, I imagined a petri dish of E. coli sitting in a slime of oil-like substance. I think I got confused with making yogurt. :-) In reality, the biofuel molecules are stored inside the cells (bacteria, in this case) and need to be taken out without harming the cells. Biofuel secretion strategies have been dubbed "milking." The difference, though, is that contrary to milk and cows, biofuels are generally toxic to the bacteria that produce them.

Several methods have been investigated to efficiently "milk" biofuel molecules out of bacteria without harming them. To understand these strategies, we need to learn a new concept: an efflux pump is a membrane transporter protein that carries a substance toxic to the cell outside the cell itself. These proteins remove all kinds of toxic substances, including antibiotics, for example, and they may be specific to one in particular, or carry a whole range.

In [1], Dunlop et al. discuss the use of efflux pumps in "milking" biofuels out of bacteria and reduce their toxicity to the cells:
"Many compounds being considered as candidates for advanced biofuels are toxic to microorganisms. This introduces an undesirable trade-off when engineering metabolic pathways for biofuel production because the engineered microbes must balance production against survival. Cellular export systems, such as efflux pumps, provide a direct mechanism for reducing biofuel toxicity."
The researchers first looked at the whole genome of E. coli to identify all genes encoding efflux pumps. They found 43 different pumps expressed in the E. coli genome, and tested them against a range of possible biofuels. Their strategy was as follows: the grew a culture of pooled bacteria with different subpopluations, each subpopulation expressing a different pump. In the absence of toxic biofuel-like substances, all subpopulations grew in equal proportions, and none had an advantage over the others. When a substance was introduced, the subpopulations with the most advantageous pumps with respect to that particular substance outgrew the rest.

This is what happened, for example, when they introduced geranyl acetate:
"When the pooled culture was grown in the presence of an inhibitory biofuel such as geranyl acetate, some efflux pumps conferred a distinct advantage. Although all strains started out with equal representation, after 38 h the population composition changed, with cells containing the advantageous pumps becoming an increasingly large proportion of the population. The efflux pumps that enhanced tolerance to geranyl acetate originated from a variety of hosts and include both known and previously uncharacterized pumps."
In their study, Dunlop et al. used a type of membrane transporters called "RND," which are made of big molecules and are only found in Gram-negative bacteria. In a more recent paper [2], Doshi et al. studied a broader set of pumps called ABC, ATP-binding cassette:
"Unlike RND proteins, transporters belonging to the ATP- binding cassette (ABC) protein family are widely found in all five kingdoms of life. They share a conserved structural architecture and specifically import or export a wide variety of molecules and ions across cellular membranes."
Doshi et al. tested whether this family of broadly specific pumps could efficiently mediate the secretion of four different biofuel molecules. Similarly to the Howard et al. paper, they used a bioengineered strain of E. coli and noticed that
"the secretion process was sustained for at least 6 days without the need to replenish the growth medium or culture. Thus, for the same quantity of biofuel produced conventionally, we have a dramatic reduction in biomass scale and significant gain in the ease of recovering the biofuel."
Though my understanding is that work still needs to be done to improve this technique and make it feasible for different types of biofuels, the fact that these transporters are spread across different species makes it potentially translatable to other organisms and therefore of broader use.

On a completely different note, can you guess what the macro picture is? :-)

[1] Dunlop, M., Dossani, Z., Szmidt, H., Chu, H., Lee, T., Keasling, J., Hadi, M., & Mukhopadhyay, A. (2011). Engineering microbial biofuel tolerance and export using efflux pumps Molecular Systems Biology, 7 DOI: 10.1038/msb.2011.21

[2] Doshi, R., Nguyen, T., & Chang, G. (2013). Transporter-mediated biofuel secretion Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.1301358110


  1. antisocialbutterflieMay 5, 2013 at 1:15 PM

    Pumps and transporters are tricky business. They can be energy expensive and tend to be picky about what they're moving. They'd need to optimize the production rate so that too much doesn't get made too quickly or the pumps won't be able to keep up. If they can find a reasonably good pump a few rounds of directed evolution might optimize the performance significantly. A decent fermentation rig might be able to pull out the media with the secreted molecule and replace it with fresh. You'd just need to keep an eye on the cell density if you aren't doing a harvest based extraction. It seems pretty feasible overall though.

    1. Hi Kat, thanks for the comment! I confess a got a bit lost when they discuss the subtleties as it is not my field of expertise, so thank you for adding those details. If you have additional references/comments with a bit more insight than my somewhat superficial discussion feel free to add them/suggest etc, thanks !!!

      For example, if they are energy expensive, does it mean you'd have to add more (insert appropriate nutrients here) to the cell cultures? I've also been wondering teh costs of producing biofuels this way, I know growing e. coli is cheap, but that's relative to what we usually need them for, whereas the case of biofuels you'd have to grow it in industrial quantities...

    2. antisocialbutterflieMay 5, 2013 at 7:55 PM

      My experience is in E. coli protein production but a lot of the principles still apply. I don't think there is one good comprehensive reference for dealing with toxic induction products. I've picked up bits and pieces troubleshooting over the years. The most productive way is to have an on switch. You inoculate the media and wait for them to reach log phase growth before you turn them "on." It's a sweet spot where there are enough cells to actually make what you want in decent quantities but not so many that they are competing with each other for the resources to make the product. If you're making something toxic it's best to slow down the process so the cells have time to cope instead of winding up with mass death.

      If what you are making is proteins they don't get out of the cell easy so you just grow a lot of low expressing cells. Since there aren't really pumps or transporters the best bet would be to slap a secretion peptide to the N-terminus if you don't want to kill off all the cells to harvest. With (relatively) small molecules the pump makes things easier as long as you don't make too much all at once.

      The energy expense is in ATP which makes the pump go. Extra nutrients can be used to supercharge the process but that is product dependent. Even rich media with extra nutrients is pretty cheap compared to most production costs. The large vat fermentation technology exists already. You just have to keep an eye on the CO2 levels, pH, and cell density to keep things in log phase and siphon off the exterior media and replace it with new. Industrialization would be pretty easy.

    3. Thanks Kat, that's super informative! So basically you are saying that if all these challenges are overcome, then costs and mass production would be downhill... If I understand your reply correctly, this is excellent news!

  2. I think I followed this and your previous post, Elena. It's like being back in college. But thanks, Doc, it's good to learn something entirely new. You know my Air Force background, so my concerns would be non-scientific:
    - Who's paying for the research and who will hold the patent?
    - Do E. coli fuels actually produce the same or nearly the same "calories" (there may well be a better term) as an equal quantity of, say, jet fuel? That is, if I can fly ten miles on ten pounds of JP-8, how many miles can I fly on ten pounds of this alternative fuel?
    - The Services are trying to be smart about alternative fuels as this Reuters article from last summer outlines:

    1. antisocialbutterflieMay 5, 2013 at 8:15 PM

      I might be able to help some here.
      (1) The paper will say who is funding it. Assuming the work isn't funded by a private organization the University always holds the patent. If it is privately funded there's usually a some sort of licensing agreement in place prior releasing the money.
      (2) Chemicals are chemicals. Assuming they are making the same molecule it won't matter whether it's made in bacteria or pumped from the ground. It all explodes the same. If they are making a less efficient combustive molecule then less caloric output. Basically it depends on what JP-8 is made of and whether it can be engineered out of a biological source.
      (3) The DOD is the best funding agency for high-risk experimental projects. It wouldn't surprise me if they've got their fingers in this some way or another.

    2. Mike, here's the info on the funding:
      "This work was supported by a grant from Shell Research Ltd and a Biotechnology and Biological Sciences Research Council (BBSRC) Industry Interchange Partnership Grant." So I guess Shell would share the patent in this case?

      As regarding the calorie question, I agree with Kat, if they produce the same molecules, it will be the same amount. I think the most pressing question is, all other parameters such as calories kept equal, how much are the costs? To be really a biofuel, in the sense that it has a lesser carbon footprint than other fuels, it would have to be more cost-effective.

  3. PS: your macro looks like the legs or arms of a doll made of tissue paper that collapse until you hang the doll up for a party. Nice colors!

    1. Haha, thanks Mike, I hadn't thought about that, but you are very close: it's sticky notes folded in that pattern and stapled. Glad you like it. :-)


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