Disaster may breed opportunity for biotech startup's oil-hungry bugs

If you can't engineer a better oil-eating bug, can you evolve one?

Scientists have long been thwarted in their efforts to create bacteria that can degrade the cocktail of chemicals in crude oil. Splicing in one foreign gene after another, they have designed microbes that, in the lab, could chew through oil's complex chemical chains. But the minute the bugs are introduced in field tests, they are outcompeted by native bacteria.

Now, one biotech firm says scientists have been doing it wrong. It is time to do some evolution.

For the past decade, a team of Florida-based biologists has been seeking to revive what has been a long-neglected field called directed evolution. Leave behind genetic engineering for a moment, they say, and instead optimize bacteria by using their own rapid evolution. All it takes is their proprietary reactor and time to train successive generations to thrive in conditions like the oil-polluted waters of the Gulf of Mexico.

Directed evolution has many possible applications -- pesticides, for one -- but it is ideal for improving the microbes that naturally digest oil, said Thomas Lyons, a former biochemist at the University of Florida and the chief scientist of Evolugate, the biotech startup. Simply put, designer bugs cannot hack it, he said.


"There's no adaptation process. The microbes are not suited for the environment they're put in," Lyons said. "They're grown in a lab. This is the fundamental problem."

Of course, Evolugate's bacteria also come from the lab. But the comparisons stop there, it says. By solving a seemingly simple but intractable lab problem, Evolugate's secretive bacterial reactor allows a theoretically unlimited number of microbe generations to evolve to tailored environmental conditions. Unfit species die, and the best oil consumers survive. Repeat, ad infinitum.

"We have a technology that we know can be applied to help this problem," Lyons said. "We know it. We're not waiting for funding or collaborations to do it. We're just going ahead."

While Evolugate has published several peer-reviewed papers detailing its machine's applications, the Gulf of Mexico spill is the company's first high-profile chance to prove itself. Five weeks ago, it began growing bacteria samples from the spill, scooped out by an employee. If it is successful in developing bugs more robust than the Gulf's natural processes -- a big if -- any product would not be ready for six months, Lyons said. The oil, though, will linger in the Gulf for much longer than that.

Nearly all scientists, including Evolugate's, agree there is no silver-bullet cure for the Gulf oil fiasco. And, being a relatively unproven startup, Evolugate's viability is highly speculative. Others have pursued directed evolution, and many small companies have made promises of solving the spill with suites of existing bacteria, treatments that scientists say would do little to nothing to clean up the oil (Greenwire, June 25).

The crucial difference for Evolugate is the selective pressure it places on microbes that are already filling ecological niches in the Gulf. They are proven bacteria, and under the machine's direction they have no choice but to improve their oil-eating abilities.

"In our machine, there is only oil to eat," Lyons said. "If the bugs don't eat oil and eat it quickly, they go extinct. This is very strong selective pressure, which speeds up the pace of evolution."

Avoiding 'GMO' label

The company has been slowly introducing itself since 2007 after its device, invented by Eudes de Crecy, Evolugate's CEO, received patent protection. (In a homage to the startup's origins at the University of Florida, the machine is unfortunately dubbed the "Evolugator.")

Applied Microbiology Biotechnology published Evolugate's first proof of concept, evolving a soil microbe to adapt to the deletion of an essential gene, in 2007. The second paper, on training a fungus to survive at higher-than-normal temperatures, came out in BMC Biotechnology last year.

In these papers, what Evolugate is proposing is not a catch-all revolution for microbial science. Its technology will not supplant or likely dent the huge steps that have been made over the past four decades in engineering microorganisms, which form an important strut of the biotech economy. For example, one of the world's largest makers of industrial enzymes, Denmark's Novozymes, develops many of its proteins from genetically engineered bacteria.

But technologies like Evolugator -- and the Genetic Engine, a French competitor that has not quite gotten off the ground -- may give companies freedom to sell optimized bacteria that, since they do not carry the burden of the "GMO" label and regulations, can be more freely applied in the wild, said Jim Spain, a bioengineer at the Georgia Institute of Technology and longtime expert in bioremediation.

"The real strength of the Evolugate approach is that they don't do genetic engineering, so there are no regulatory barriers to using the organisms," Spain said. "It's very sophisticated, a rapid strategy for evolution of a bacteria."

Spain, who serves on Evolugate's scientific advisory board, began a collaboration with the firm in 2006, testing its machine's ability to improve the yield of wild microbes. While those experiments focused on natural bugs, Spain suspects in the end that directed evolution will be combined with genetic engineering, which often weakens bacteria while giving them foreign abilities.

"The combination of the two would be very powerful," he said.

The tandem could provide an answer to scientists who have long sought to deploy bacteria to treat oil spills, a process known as bioaugmentation.

The field received huge amounts of funding and interest in the 1980s and 1990s, as companies like General Electric Co. explored genetic engineering as a way to clean PCB contaminants from the Hudson River, said Terry Hazen, a bioaugmentation researcher at the Lawrence Berkeley National Lab, in a recent lecture.

"A lot of companies thought, 'Oh, well, we could patent a bug and this would be great.' And what we're actually finding out is that it's not necessary," Hazen said. "In fact, it doesn't offset all the costs for making the bug. Quite often the bug dies and ends up becoming a nutrient source for the indigenous bugs."

Scaling the 'wall'

The directed evolution of bacteria is nothing new.

During the 1950s and 1960s, it was the bleeding edge of biology. Scientists designed reactors, called chemostats, that provided sealed, sterile environments for growing bacteria at a near constant rate. By partially flushing the chamber and changing nutrients and environmental conditions, successive generations of microbes evolved.

But soon enough, chemostat-mediated evolution waned in popularity, Lyons said. After the discovery that DNA sequences could be manipulated, biologists feverishly sought to edit DNA, resulting in breakthroughs like recombinant DNA, gene sequencing and, recently, the first synthetic lifeform.

"People just turned to genetic engineering because it was easier and it was sexier," Lyons said. But after a few decades, he said, people began "realizing there are certain traits you can't access in genetic engineering. ... How do you change the ability of a microbe to grow at a higher temperature? We just don't have the knowledge to do that."

While genetic engineering came to dominate the field, chemostats had also, quite literally, hit a wall. As bacteria become accustomed to reactors, they begin devising alternate survival strategies by sticking to the walls of the reactor, hanging on when the vessel is diluted. And while wall-adherence is an admirable trait for the bacteria, most industrial producers are after qualities like growth rate and yield.

Partially because of this "wall growth" problem, most current implementations of directed evolution are heavy on the manpower of underpaid graduate students, using a process known as serial dilution that transfers bacteria cultures to new medium on a daily basis. While outwardly inefficient in its high maintenance, it is also quietly inefficient: Often, only limited samples are transferred from one culture to the next, meaning many beneficial mutations are missed.

Put simply, Evolugate has solved the wall problem, it says, by using flexible plastic tubing as the culture chamber.

With each automated cycle of the 6-foot-tall machine, the tubing and medium are changed in a belt-driven process powered by peristaltic movement, mimicking how food is passed down the esophagus. It is difficult to visualize, but it results in a "machine that runs itself" with only basic monitoring required, Lyons said.

Into this machine, Evolugate put a slop of dispersed oil and water pulled off the Gulf's surface south of the Mississippi River Delta. The mix, including native microbes and a supplement of 18 oil-degrading bacteria, was set for the Gulf's hot summer surface temperatures and then left alone. At first, many microbes died as a result of a host of variables: temperature, salinity, oil components, dispersants and marine viruses. But after a week, the lab began to see strong growth.

Five weeks on, Lyons has seen the evolved bacteria thrive against its controls, though any excitement should be held until the company can prove its bugs in the field, where many previous augmentation promises have gone to fail.

"What we see is a clear improvement in the performance of our community from week three to week five in terms of final cell density and growth rate," Lyons said. "Evolution is working. They are eating more and eating faster."

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