Markus Ribbe and Yilin Hu: Brewing biofuel from bacteria

Faculty Spotlight
August 17, 2017 By Wendy Wolfson

In a high-ceiling room in professor Markus Ribbe’s lab, a giant still emits pungent vapors from fermenting bacteria. Ribbe, chancellor’s professor of molecular biology and biochemistry at the UCI School of Biological Sciences, and his colleague and wife Yilin Hu, assistant professor of molecular biology and biochemistry, are brewing fuel. They are harnessing nitrogenase, an enzyme derived from a common soil bacteria, to create hydrocarbons from carbon monoxide and carbon dioxide. The goal is to produce an affordable, truly renewable biofuel, as well as essential industrial products.

Brewing up a better biofuel

The world economy still relies on carbon-based fossil fuels. Developing renewable alternatives that can compete with the cheap cost and energy density of fossil fuels is one of the biggest challenges of our time. Continued use of fossil fuels is unsustainable because of their steep and growing environmental costs, and for strategic reasons as well. Newer generations of alternative fuels are becoming more efficient and cost-effective to produce, but they still have technical and scale-up issues, as well as their own environmental costs. Currently, the main gating factor hobbling innovation in alternative fuels is the relatively low price of oil, but that will not always be the case.

Ribbe terms his lab’s bacterial fuel factory concept a “fourth generation” solution. The discovery came about quite accidentally. Ribbe, a microbiologist, did his Ph.D. on microbes that can use carbon monoxide as a feedstock. In 2010, his lab was researching the properties of nitrogenase, a catalyst produced by Azotobacter vinelandii, a “completely harmless” common soil bacteria that has the ability to affix nitrogen in the soil to plant roots. Nitrogenase is a metalloenzyme, meaning it is metal based; the enzyme can be molybdenum or vanadium-dependent. The nitrogenase enzyme family catalyzes the reduction of nitrogen to ammonia, an important step in the global nitrogen cycle.

In 2010, Ribbe and his students were performing some simple kinetic experiments with an enzyme variation of nitrogenase. “We added carbon monoxide, which has always been believed in the field to be an inhibitor of ammonia formation,” Ribbe said. “The electrons got sucked up by carbon monoxide.” When the students injected some of the enzyme mixture in a gas chromatograph, they saw that it produced ethylene and propane. “At this point none of us believed it,” Ribbe said. So they did another experiment, labeling the carbon atoms with a specific isotope. Under the chromatograph they found the labeled carbon atoms in the ethylene and propane. “We were not really looking for this,” Ribbe said. “I would say it was a side discovery which was really the best of our lab.”

Ribbe and his students found that this nitrogenase enzyme family could catalyze the reduction of carbon monoxide (CO) or carbon dioxide (CO2) to produce certain hydrocarbons under moderate conditions: normal pressure and ambient temperature—without requiring hydrogen; it can also use waste feedstocks like carbon dioxide and carbon monoxide to do so. “A very important aspect is that it is very environmentally friendly,” Ribbe said.

A bacterial fuel factory?

Ribbe hypothesized that the ability to reduce carbon monoxide could be an evolutionary relic of this particular enzyme family, and perhaps, this ancestral nitrogenase could be a link between the carbon and nitrogen cycles on earth. But whatever its antecedents, this peculiar property of Azotobacter vinelandii could potentially be harnessed to generate fuel and essential industrial products.

Currently, most hydrocarbon products for energy or the production of industrial plastics and rubber—including methane, ethylene, propylene, propane and butane—are synthesized via the Fischer-Tropsch process, a series of chemical reactions that converts a mixture of carbon monoxide and hydrogen into liquid hydrocarbons and requires high temperatures and pressures, and lots of hydrogen to work. Hydrogen is energy-intensive to synthesize, requiring fossil fuels. The Fischer-Tropsch process makes an excessive amount of the greenhouse gas methane as a waste product.

Another essential industrial process, the Haber-Bosch method, is an artificial nitrogen fixation process. Using high temperatures and extremely high pressure, this process combines hydrogen and nitrogen derived from air over a catalyst to produce ammonia, critical for the fertilizer that enables modern large scale agriculture—and warfare. The Haber-Bosch method also requires a lot of energy, high temperatures and high pressures to work.

Next step: Scale-up and industrialization

Ribbe envisions growing the Actinobacter vinelandii bacteria on industrial carbon waste to generate hydrocarbons. He and Hu found that the A. vinelandii strain that expresses the nitrogenase enzyme can produce methane (CH4), ethylene (C2H4), ethane (C2H6), propylene (C3H6), propane (C3H8) and butane (C4H10).

So far Ribbe and Hu have optimized the bacteria to pump out the catalyst in greater amounts. Their lab is now engineering the enzyme to make longer-chain hydrocarbons for liquid fuel, with the goal of getting seven to eight chain carbon products. “Ideally, if you could get carbon monoxide and carbon dioxide as feedstocks, you could make them convert this into hydrocarbons,” Ribbe said. The researchers showed that nitrogenase can convert carbon monoxide and carbon dioxide into products like propane and butane. “This enzyme catalyzes at moderate conditions, room temperature and ambient pressure, and doesn’t need hydrogen,” Ribbe said.

Currently Ribbe and Hu are ramping up a process conducted in small flasks to a pilot bioreactor of perhaps five liters, and running continuous cultures to figure out how long it takes to produce hydrocarbons efficiently.

According to Ribbe, the next steps towards eventual large scale production of fuel and industrial products will be optimizing the reaction and developing bioreactors that can hold large quantities of immobilized nitrogenase enzyme, eventually leading to the manufacture of reactors driven by catalysts that mimic the action of the active site of the enzyme.

“What we envision is to build up a bacterial system where we can introduce an enzyme to carbon monoxide that is feedstock-free,” Ribbe said. “Bottom line, it is fairly different compared to other approaches to make biofuels.”

“It is amazingly simple,” Ribbe said. “At this early stage it works at low rates, 1% to 2% conversion of carbon monoxide.” Ribbe noted that the enzyme does this as a secondary reaction. To derive products such as propane, butane and ethylene, it is not necessary to do lipid extraction as with algae-based biofuels, which will save a step in production.

Scalability of the process to mass production will be the biggest challenge. “So far the catalytic numbers are fairly comparable to some Fischer-Tropsch methods,” Ribbe said. “One question is, how efficient is the catalyst? Industry wants a full conversion of substrate. While the Fischer-Tropsch process does a full conversion, our enzyme currently does a low percentage of conversion at a comparable efficiency.”

“I think this is really a different approach compared to other process that are out there,” Ribbe said. “The first generation, second generation, all other approaches are limited to fermentation processes. This system can directly convert carbon monoxide or carbon dioxide to hydrocarbons without going through a fermentation process. That is one big advantage.”