Fishing for bacteria: A step into the biofuel world


By Travis McKnight,

The United States is moving increasingly closer to an oil shortage shock that will make the 1973 fuel fiasco, in which prices quadrupled in mere months and people stood in line for hours to fill up a portable gas tank,  look like a child’s game. The National Intelligence Council has predicted soaring costs for food, water and energy within the next 15 to 20 years. Alongside these pressing matters are global overfishing and climate change. If the status quo is maintained, mankind’s future is going to be pretty bleak. The need for renewable, alternative resources for energy is more important than ever.

Renewable forms of energy have progressed over the last decade, particularly solar power and electric vehicles. Solar energy has reached the point where U.S. utility companies predict homeowners with solar panels will actually bankrupt utility companies, and electric vehicle manufacturer Tesla has become popular after creating an all-electric sedan that can travel 300 miles between charges. While these advancements are a step in the right direction, they require a large consumer investment —one in today’s global economy many cannot afford.  This is where bacteria-based biofuel comes into the picture.

There is an enormous potential for biofuels to shape the energy market. Unlike oil drilling and fracking, fuel creation with cyanobacteria is environmentally friendly, and can be used as diesel fuel and food while simultaneously acting as a carbon sink. If perfected, the technology will able to tackle three problems at once: fuel scarcity, food shortage and climate change.

But to accomplish the food supply and carbon offset, cyanobacteria’s fuel possibilities must be tapped first, since everything else is a byproduct of the production process. However, unlike ethanol diesel fuel using corn, bacteria are pesky organisms that quickly adapt to their surroundings and can be difficult to tame. This has presented researchers with unique sets of challenges and opportunities.

Researchers at Arizona State University are working with a cyanobacteria strain, called synechocystis pasteurized cyanobacteria collection-6803, with the objective of making marketable cyanobacteria-based biofuel that challenges petroleum-based energy. So far, the university has seen moderate success in its experiments.

In 2011, the three ASU laboratories tacking this issue reached a breakthrough by genetically modifying synechocystis to produce and secrete free fatty acids (laurate) in a contained environment without harvesting the organism. This process allows the cyanobacteria to work as a self-contained factory with multiple lifecycles because it’s no longer necessary to break open the bacterium’s cells and extract the materials, which was costly in time and energy.  However, this discovery illuminated new problems for the teams to work on.

The dilemma that arose, which researchers are currently focusing on, is that the large scale 4,000 liter photo-bioreactor is susceptible to invasion by foreign bacteria that will eat the laurate synechocystis produces. The rooftop reactors are in an aseptic condition, in which 99 percent of the solution is cyanobacteria, but one percent is outside heterotrophic “scavengers” that made it into the solution. That one percent is thought to be impossible to prevent, and it alone consumed all the laurate synechocystis produced.

Survival of the fittest

There are five commonly represented invading heterotrophs isolated from the photo-bioreactor (Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Bacilli and Actinobacteria), and all of these eat laurate incredibly quickly — an entire batch of free fatty acids in a 4,000 liter reactor can be devoured within seven or eight hours. Although these five species are the common invaders, they are not always present in every batch.

One theory regarding which bacteria strains will invade a culture is akin to a competitive lottery. If a group of bacteria with similar metabolic structures have the capacity to colonize an entire reactor’s culture, out of that group, only one or two of those strains will randomly be able to complete the colonization. Since all of these bacteria grow with bacteria that have similar metabolic capacity, it’s likely that they are related taxonomically. Out of this large taxonomic bracket, it’s possible to discover which bacterium wins the random colonization in one tank, and which one wins in another. This leads to clues as of why one succeeds while another fails even though reactors are treated the same.

Alex Zevin, a third year Ph. D. student focusing on biological design who has worked for the ASU biofuel lab project since summer of 2011, wants to rig the lottery with a genetically modified heterotroph so the winner is chosen from the beginning, and every other bacteria starves to death.

The cyanobacteria produce an abundance of organic materials instead of just the fatty acid; namely, lots of sugar and protein, which is all that’s needed to live, on a simple metabolic level. The starvation approach is to introduce a probiotic that is going to eat everything in the culture besides the fatty acid, and hopefully this non-fatty acid consuming heterotroph will outperform every other invader to the point where they have no nutrients to devour.

Let’s say in the substrate there are piles of sugar, proteins and fats, which is basically what synechocystis produces in the reactor. The sugar is immediately available energy, and that’s what all the heterotrophs are there to get. The protein is auxiliary, it’s going to be there no matter what, but it’s not as easy to eat. The fat is super high in energy, but it takes a lot of effort to get the energy. Zevin is trying to find a bacterium that will eat up all of the sugar and protein right away, so two important substrates are out of the reactor, and now all that’s left is the fatty acid. However, a problem still remains in actually finding a bacterium that will eat everything but the fatty acid.

Building a better bacteria

Zevin’s idea is to remove the beta-oxidation pathways of the isolated heterotrophs, and make it so the bacteria physically can’t eat the laurate. Beta-oxidation is a metabolic process in every living organism where fatty acids are broken down to be used as energy.

His current theory is to isolate one of the five commonly found heterotrophs, genetically remove its beta-oxidation pathways and then take that modified strain and put it back into the culture. If it’s growing, but can’t eat the fatty acid, it must be eating other nutrients.

In order to observe the before and after growth rates, Zevin must discover how much fatty acid the isolate eats in its original state. By introducing the heterotroph into a flask of fatty acid at various concentrations, the rate of consumption and growth can be measured. After this process is completed, a predictive model can be built that basically shows the maximum growth rate, and how much substrate will be left over. With these tests Zevin says he hopes to discover how to exclude certain bacteria.

Let’s say the tests show a particular bacterium is commonly winning the competitive lottery and it thrives at a certain level of substrate, but doesn’t do well below that amount. The goal can then become to keep the substrate concentration at the lower base level so the heterotroph can’t efficiently survive. This process is repeated with different bacteria and different substrate concoctions.

However, the five isolates are not well-studied in comparison to some bacteria, such as e-coli. Because of this, it can be difficult to study why the heterotroph is reacting in certain ways.

Zevin also plans to buy a batch of e-coli from the Internet that already has its beta-oxidation pathways knocked out and then introduce that strain into the culture. Because e-coli strains are incredibly studied this process will allow gene expressions to be investigated, which makes its growth traits observable. Nonetheless, because a validation test hasn’t been performed yet, this entire theory could go down the drain.

In any ecosystem there are multiple niches that fill different roles, and that is one fear Zevin has about the photo-bioreactor. It’s possible that so many diverse communities are present is because there isn’t a singular heterotroph eating all the sugars, proteins and fatty acids. Contrarily, there could be one bacteria eating sugars, and another eating fatty acids and then a different one eating the protein. “Hopefully it’s not a never-ending road down the food web,” Zevin says. If the ecosystem model comes to fruition, the method of countering it likely wouldn’t differ from isolating and modifying unique strains; the community will simply be built one bacteria at a time.

“I think we’re kind of on a fishing trip right now,” Zevin says. “Often it’s an insult to refer to a scientific work as a fishing expedition, but that’s what we’re doing. I’m out there trying to catch the right guy at the right time.”


Travis McKnight is print and multimedia journalist with a passion for discussing science, technology, the environment and video games. He recently graduated from The Walter Cronkite School of Journalism and Mass Communication at Arizona State University, and his exploits and random ramblings can be followed on Twitter @Khellendos.