Microbes from a lake in Berkeley, California’s Tilden Park cracked the mystery of how microbes produce a key component of gasoline.

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In 1996, scientists found a fuel-producing bacterium at the bottom of a Swiss lake. Now, they know how it works

Twenty-two years ago, microbiologists in Switzerland stumbled on a mystery deep in the muck of Lake Au, an offshoot of Lake Zurich: a bacterium that naturally produced a component of gasoline called toluene. Now, researchers have discovered how some bacteria manage to make the toxic hydrocarbon. Why they do so remains a puzzle, however.

“This is a really nice piece of science,” says Alfred Spormann, a chemical engineer at Stanford University in Palo Alto, California, who was not involved in the work. “I think it’s a terrific example of using metagenomics and biochemistry to learn about organisms that are difficult to study.”

The Lake Zurich discovery fingered a bacterium, Tolumonas auensis, as the toluenemaker, and raised the prospect of culturing it to produce the hydrocarbon as a fuel. T. auensis isn’t the only bacterium that makes hydrocarbons, but its byproduct is odd: Toluene is packed with energy, meaning an organism must expend a lot of energy to produce it. It’s also toxic. But T. auensis is particularly hard to culture in Petri dishes and study with the normal tools of molecular biology. The trail ran cold.

A few years ago, Harry Beller, an environmental microbiologist at the Joint BioEnergy Institute (JBEI) in Emeryville, California, decided to reopen the case. His team first tried recreating the 1996 experiment, in which researchers got T. auensis to make toluene. But after multiple attempts, Beller’s team struck out. So, he called Friedrich Jüttner, the group leader from the original Nature paper, to ask for advice. Jüttner, Beller says, told him not to worry about T. auensis and suggested that he could probably find a similar organism doing the same thing in anoxic sediments from any nearby lake.

So Beller and his colleagues drove 13 kilometers from JBEI to Tilden Park in nearby Berkeley, where they took samples from a small reservoir called Jewel Lake. Back at the lab, they found that Jüttner’s words were prescient: After culturing the microbial communities, the lake sample registered traces of toluene. They found similar traces in samples from a nearby sewage treatment plant.

To sort out what the bugs were doing, Beller and his colleagues first turned to the sewage sample. They harvested and broke apart the bacteria, collecting all the proteins. They then divided these proteins into successive fractions, winnowing out those that showed no signs of producing the hydrocarbon. They ran the “keepers” through genomic scans, creating a library of more than 600 candidate genes from the two samples. Previous analyses by Beller and colleagues had suggested that the genes responsible for making toluene were likely glycyl radical enzymes (GREs), a small family of proteins that carry out other challenging chemical reactions. They always come paired with an activating enzyme. So Beller’s team looked for just such a cluster of genes among the candidate genes in the toluenemaking sewage sample, and they found the GRE phenylacetate decarboxylase (PhdB) and its activator, PhdA. “We looked for it in the lake culture and found it there, too,” Beller says.

To confirm that these were the toluene-producing proteins, Beller’s team transplanted the genes into easily cultured Escherichia coli bacteria, which expressed the proteins. The researchers purified the proteins and added them to a vial containing phenylacetic acid, the normal starting material for PhdB enzyme. But in this case, the phenylacetic acid was made using carbon-13, a rare isotope of carbon that enabled Beller’s team to trace the fate of the compound as it reacted. The researchers found that the enzymes produced C-13–labeled toluene, they report this week in Nature Chemical Biology. The result confirms the enzymes are the ones at work in the muck.

So why do microbes produce toluene? They might use the toxic compound to ward off competing bacteria, Beller says. A more likely explanation, he suggests, is that the production of toluene provides a strategy for the bacterium to regulate its internal pH in a somewhat acidic environment. The toluene-producing reaction consumes protons in the cytoplasm—the gooey fluid in the cell—and thereby can lead to an increase in the pH of the cytoplasm. This can protect against the more acidic conditions that likely exist in the cell's environment, such as anoxic lake sediments or sewage sludge.

Whatever the explanation, Beller says he and his colleagues are now working to engineer other easily cultured organisms to make toluene. Eventually, that could lead to industrial microbes that synthesize one key component of gasoline. But because toluene derived from oil is cheap, even perfectly functioning bugs may not find much of a market. Solving a mystery may be their work’s biggest payoff.

*Correction, 27 March, 10:15 a.m.: An earlier version of this story contained several factual errors. The explanation for why bacteria may produce toluene, a component of gasoline, has been revised to reflect the fact that it may help particular anoxic bacteria regulate their pH.