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

Fill the tank, please. With bacteria!

I apologize if you've already heard about this, but the paper is really cool and I couldn't resist discussing it here.

Escherichia coli, or E. coli for brevity, is a bacterium normally associated with "bad" things like food poisoning. Even though most strains are actually harmless, even the CDC has a page dedicated to E. coli outbreaks. Since it's part of our gut flora, the lower intestines in particular, it's usually not a good sign when E. coli is found in places like restaurants and cafeterias. (Yuck!)

What's less known to the public is that E. coli is one of the most studied bacteria and makes a great model for mutations, gene duplications, and horizontal gene transfer.

What's even less known is that this amazing bacterium has the potential to save our planet from further drilling. How? By producing fuel. Yes, you read that right: through a combination of gene modifications, researchers from the University of Exeter [1] induced "petroleum-replica hydrocarbons" production in E. coli. These hydrocarbons are structurally and chemically similar to fossil fuels.

In their paper, Howard et al. argue against the current biofuels because they bring additional costs in downstream processing and are not 100% compatible with the engines on the market.

"To overcome the end-user blend wall, it is essential to generate precise chemical replacements to fossil fuels through sustainable means.Retail transport fuels are composed primarily of hydro- carbons (n-alkanes) of various carbon chain lengths (Cn), branched hydrocarbons (iso-alkanes), and unsaturated hydrocarbons (n- alkenes). The ideal biofuels are therefore n-alkanes, iso-alkanes, and n-alkenes that are chemically and structurally identical to the fossil fuels they are designed to replace [1]."

Gasoline, diesel and jet fuels are made primarily of molecules called alkanes, or saturated hydrocarbons. Most people are familiar, or at least have heard of methane, the simplest alkane molecule. These molecules are naturally produced not just by bacteria, but also by plants and insects when they metabolize fatty acids. In 2010 Schirmer et al. described in a Science paper [2] an alkane biosynthesis pathway in cyanobacteria, commonly known as blue-green algae.

"The pathway consists of an acyl-acyl carrier protein reductase and an aldehyde decarbonylase, which together convert intermediates of fatty acid metabolism to alkanes and alkenes [2]."

Understanding how alkanes are produced and, in particular, which genes are involved in their production, was the first step. The second step was answering the question: can we tweak this pathway to produce alkanes that can replace our current fuels?

Seen under this light, the PNAS study published last March 15 [1] is a bioengineering success story. Howard et al. designed a novel metabolic pathway that forced E. coli to use free fatty acids instead of fatty acid compounds as in cyanobacteria, and produce fuel-like alkanes, what the authors call "industrially relevant, petroleum replica fuel molecules." Once finalized, this type of biofuel will be compatible with current engines and will not need to be blended with other petroleum derived chemicals.

A bit of perspective: though derived from natural and biological sources, biofuels still contribute to pollution, carbon emissions, and global warming. Despite the amicable "bio" prefix, they all come with a non-null carbon footprint, some more than others. The true efficiency of any kind of fuel is the energy they produce minus the energy and costs it takes to derive them. For example, producing biofuels from crops drains precious resources, first and foremost, water, but also arable land, forests when arable land is not available, and food sources in underdeveloped countries.

So here's where biofuels from bacteria have a striking advantage: E. coli is one of the cheapest and easiest bacterium to grow in a lab. It doesn't drain water reservoirs and it doesn't need deforestation to grow. Contrary to most biofuels out there, that have high production and energy costs, the carbon footprint of biofuels derived from bacteria only comes from carbon emissions when you burn them.

And while this is an excellent thing, I still think that the real change we need to make to preserve our planet is to switch to renewable energy.


[1] Howard, T., Middelhaufe, S., Moore, K., Edner, C., Kolak, D., Taylor, G., Parker, D., Lee, R., Smirnoff, N., Aves, S., & Love, J. (2013). Synthesis of customized petroleum-replica fuel molecules by targeted modification of free fatty acid pools in Escherichia coli Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.1215966110

[2] Schirmer, A., Rude, M., Li, X., Popova, E., & del Cardayre, S. (2010). Microbial Biosynthesis of Alkanes Science, 329 (5991), 559-562 DOI: 10.1126/science.1187936

Green living

Though the main focus of this blog is genetics and science, I hope you'll allow me a slight change of topic today. I am honored to have as a guest blog my friend Jeff Casalina, who is an energy renewal specialist with the Department of Energy. Jeff is currently working on a book proposal on energy and transportation fuel renewal, and has kindly accepted to tell us about his vision for a greener and planet-friendly way of living. I hope you will enjoy what he has to say. It's something we can all promote and contribute to.

Question:   Of the following life necessities—food, water, air, clothing and shelter—which is your most immediate need?  Answer:  Air, of course!  But, what is your second most pressing need?  (Hint:  think sub-zero temperatures.)  You guessed it…shelter!  You might be hungry and thirsty, but when you are stuck in a raging snow storm or tornado, shelter is your immediate concern. 

We think of our shelter on so many levels—protection from elements, comfort, security, prestige, investment—but what about sustainability? 

Sustainability in housing can be defined in several amenities.

Simplicity-  How many times have you been on vacation and rented a small cabin or cozy hotel room and thought, “I could really live in something like this.  I don’t really need the big house and all that stuff.”   Simplicity equals low cost and energy efficiency.  The smaller the house, the less resources, heat, light, water and impact to the environment. 

Energy Efficiency-  The “low hanging fruit” of the fossil fuel tree has already been picked.  Regardless of what you have heard about oil shale and tar sands and vast deposit of natural gas, what are left are small pockets, remotely located and/or stuff requiring costly or environmentally damaging extraction methods.  (Please view the documentary, Gas Land.)  It’s time we get off it.  Every house has the potential to generate all of its own energy.  Solar water heating, solar and wind generated electricity are all common technologies, and every house has enough roof area to accommodate sufficient solar panels.   Energy efficient appliances and lighting reduce demand.

Design-  All new construction should incorporate passive solar design and earth sheltering.  Passive solar design uses south-facing windows to accept the sun’s rays.  Dark colored, high mass materials absorb the sun’s warmth during the day and re-radiate it at night.  A properly designed overhang keeps the high summer sun off the windows.  Earth sheltering, particularly on the north side, protects against cold north winds and provides basement-like coolness in summer.

Water Efficiency-  All new construction should incorporate water conservation.  A possible water conservation feature is separate gray water (shower and bathroom sinks) and black water (toilets and the kitchen sink).  Gray water can be used to water your garden.  Other water saving features include: waterless urinals and dual flush toilets (one button for number one, the other for number two) low-flow showerheads and rooftop catchments systems.  Every house, even in New Mexico, should be able to capture enough usable water.

 Impediments to sustainability are exuded by our consumer culture.  We are constantly bombarded by advertising that implies that bigger is better, and fancier is sexier.  Resist!  Turn off the TV.  Read and educate yourself on the topics that will make this a better planet (and you much happier).  Collectively, our individual efforts will devolve the consumer culture.  

And if you think about it, our ultimate home is Planet Earth, the one shelter we all share. Let's all pitch in and take care of it, if not for ourselves, for our children. Just like the Native American proverb says, We don't inherit the Earth from our Ancestors, we borrow it from our Children.

Thanks, Jeff. I can't wait to read your book! 

Photo: Seattle Arboretum. Canon 40D, focal length 61mm, exposure time 1/6.