Suffocating cells for science
If you believe that all living things need oxygen to breathe, you’re not only wrong, but hopelessly human-centric. But don’t be too hard on yourself. Most mammals are biased toward multicellular organisms.
It’s true that humans, along with mammals, birds, even insects and fish, require oxygen for survival. But not bacteria. What bacteria lack in intellect, they make up for in the extraordinary adaptability of their metabolism.
On Earth some 3 to 4 billion years ago, primitive microbes thrived in the absence of oxygen — anaerobically — by “breathing” iron-based minerals. Many species of bacteria still have the ability to do this.
That’s right. Cells can breathe rock. And to do so, they must send their electrons outside of their cell bodies, sometimes moving them great distances.
Chemists understand respiration differently than the rest of us. At the cellular level, it is simply this: taking electrons from food and giving them to the thing they’re breathing, whether that’s oxygen or rock. When oxygen is involved, this is a fairly simple process. Since oxygen is a soluble molecule, it gets easily diffused from the outside to the inside of the cell through the cell membrane.
“But what do you do if the thing you’re trying to pass your electrons to, a.k.a. the thing you’re breathing, is outside the cell?” asked Moh El-Naggar, an assistant professor at University of Southern California, who runs a lab dedicated to basic research. “Somehow you have to build a bridge to get your electrons to the outside.”
At his lab, El-Naggar and his team obsess about this process: how the electron gets from Point A to Point B.
“It is a beautiful problem at the interface of physics and biology,” he said.
Unlocking the secrets of how these processes work could lead to breakthroughs in semiconductors, fuel cells, solar power and understanding how life can thrive in the harshest environments.
In May 2009, El-Naggar made a discovery, from which all of the experiments in his lab have since sprung: A few years earlier, a pair of scientists discovered that microbes grow long, hairy filaments or fibers that are electrically conductive. El-Naggar had a hypothesis. These fibers, he suspected, serve as a conductive bridge between the cell and the rock that they’re breathing. In other words, the path the electrons take to move from the cell body to material outside the cell.
Testing this theory was a two-year process, which required growing cells and building fantastically tiny rods to study the conductive properties of the fibers, or as they’re now called, “bacterial nanowires.”
It was late one night at Lawrence Berkeley National Laboratory that El-Naggar connected all the pieces, quite literally. He had flown out that morning with a silicon chip in a carry-on Canon camera case. The chip contained thousands of dead, but well-preserved cells, and their newly sprouted nanowires.
In a clean-room facility there, he used a powerful electron microscope to build dozens of tiny platinum electrodes – leads, he calls them — each measuring a micron long. That’s about 100th the width of a human hair. Then, with an electron beam, he deposited electrodes onto his chip, placing them at each end of several nanowires.
Once the nanowires and electrodes were connected, he moved the chip to a probe station containing an optical microscope and a voltmeter, fastened the chip to a plate and turned a knob to pump in electricity. He wanted to know if electrical current could move through the nanowires. And over the course of several minutes, he studied a figure on the screen.
“What you’d expect to see is that you get more current flowing through the nanowires, the more voltage you apply to them,” he said. “And that’s exactly what we saw.”
El-Naggar believed that current was generated by electrons surging through the nanowires to the platinum leads. But to be sure, he had to cut the nanowires and repeat the experiment. If he was right, breaking the fibers would kill the current. Using an ion beam — a tiny scalpel made out of pure energy — he sliced the wires. And then he performed the experiment again, hoping that he would see… nothing.
“It’s kind of weird, right?” he said. “People think we get more excited about seeing stuff. But every good experiment needs a good control. And sometimes nothing is as exciting as something.”
As he’d hoped, the numbers stayed frozen at zero. Alone in the lab, he pumped his fist in the air and then sent an email to his collaborators. One of his postdocs had vowed to grow his beard until the experiment was complete. In his email, El-Naggar recommended a shave. He had a bushy beard, he recalls now. “And it was beginning to look pretty bad.”
The finding was published in the journal PNAS in 2010.
El-Naggar now oversees a lab of five graduate students, two postdoctoral researchers and a small army of undergraduates. He was one of 102 researchers to receive the Presidential Early Career Award for Scientists and Engineers earlier this year. And his Twitter posts look like this:
Spread the word: I have an opening for a bioelectrochemistry postdoc to work on single microbe and single mitochondrion electron transport.
— Moh El-Naggar (@BioPhysicalMoh) March 31, 2014
Intimidating, right? But he is anything but.
“He’s the best advisor the physics department has had,” said Benjamin Gross, a postdoctoral candidate in his lab, who studies the basic biophysics of how electrons move. “Some advisors ignore you; some are anxious for you to publish good work,” but El-Naggar, he said, gets in the trenches with the young scientists. “People love working here.”
One big discovery, many applications
The whole story of El-Naggar’s lab since that night in Berkeley is about trying to move the ball forward on the electron transport discovery. And his team has taken that basic concept and launched it into wildly different directions of research.
One member of his team, Yamini Jangir, traveled to Death Valley, where, among the sand dunes and jagged salt flats and hellish 115-degree temperatures, she collected water samples from a giant pressurized well. In that water, bacteria thrives deep underground in low oxygen conditions.
Back in the lab, Jangir built her own reactor out of a polypropylene jar, plastic piping, titanium wires and aluminum foil. Her hope is to recreate in the lab the anaerobic conditions from the Death Valley subsurface that allow this bacteria to thrive. Statistics for these bacteria are grim now — a mere 1 percent survive in the lab setting. The ultimate goal: to develop a technique that would allow scientists to cultivate underground bacteria in a lab setting, and thus study them better.
Another graduate student, Ian McFarlane, is trying to make semiconductors in the greenest possible way – using only cells. And Benjamin Gross, a postdoctoral candidate, is studying the basic mechanism of how cells shuttle their electrons. (McFarlane and Gross both rely on a bacteria called Shewanella that was discovered in Lake Oneida in upstate New York.)
Against the wall in the wet lab is a fridge with glass bottles filled with arsenic and sulfur — nutrients for the bacteria — and a sign that says “No Food or Drink.” A “Poison” label is taped onto many of the bottles, which are sealed with black rubber caps to keep the oxygen out. A handful have a skull and crossbones scrawled across their midsection in black Sharpie.
On McFarlane’s computer screen are dozens of nanofibers, each about 15 microns long and ranging from 10 to 300 nanometers wide — roughly 1,000 times thinner than a sheet of paper. He hopes these will someday have a practical use as semiconductor devices, possibly as cheap solar panel material. The key, he explains, is having the bacteria do the work.
“They’re my little factories,” he said. “Rather than having a person make insulin, you have a bacteria make insulin. Rather than having a person make nanoscale fibers, you have bacteria do it.”
McFarlane moves to the far end of the room, where he’s done just that. He pulls a glass jar from a refrigerator. The jar contains a thick yellow substance resembling foam insulation. But when he shakes it, the consistency changes, becoming more liquid, like orange juice. The resemblance is so strong that he can no longer drink orange juice from a clear glass, he says.
Inside the jar are billions of nanofibers created by combining arsenic and sulfur, adding bacteria and removing oxygen.
“I love the smell of rotten eggs,” McFarlane said. “It means it’s working.”
Removing the oxygen forces the bacteria to use a backup power source. In other words, to breathe the arsenic and sulfur. And the nanofibers that grow during that process act as primitive semiconductors.
Magnified on his computer, MacFarlane studies the fibers. Some look like smooth rods – others sport a crystalline pattern, like a spinal cord.
He’s creating fully-functioning transistors out of these semiconductors, El-Naggar explains. Understanding their shape and structure could help engineers put the incredible metabolism of these microbes to practical use. Engineers, they hope, could one day harness the bacteria’s breathing system to power fuel cells, for example.
So many dead cells
A few feet from McFarlane, undergraduate James Lu painstakingly prepares slides for Gross, who studies Shewanella bacteria cells one at a time as they pass their electrons onto electrodes. They are clumsy-looking slides with black and white wires sticking out, cover glass embedded with electrodes, and a plastic chamber, mounted onto a plate with double sticky tape.
On his computer, Gross pulls up a graphic of a cell. The cell takes in food, he explains — acetate or lactate, for example. It converts the food into something called ATP – Adenosine triphosphate. And then it spits out the electron.
“It needs to get rid of the electron, or it will gum up the works,” he says.
Gross moves to the lab counter, and using a syringe, draws half a milliliter of solution from a glass bottle and injects it into the chamber on the plate. Then he caps the two ends so they’re airtight. He has just injected millions of cells into the chamber.
You can’t see them now, but the glass on the plate is patterned with electrodes made of indium tin oxide, a material that’s widely used in smartphone touch screens, solar cells and as a defroster for Boeing 747 aircraft windshields.
Earlier, he had bubbled nitrogen into the solution and added the amino acid, cysteine. Both work jointly to starve out the oxygen, so the cells will be forced to breathe the ITO, just like MacFarlane’s cells were forced to breathe sulfur and arsenic.
“We’re suffocating the children,” Gross says, and impersonates the bacteria. “Fine. I’ll breathe your electrode.”
In a dry lab across the hall is a device called an optical trap that Gross spent six months building. It relies on an infrared laser to trap and isolate cells, a laser that could blind you, he warns, causing excruciating pain if your eyes were to wander directly into the light without safety glasses.
“There. I found an electrode,” he says. “Now we kind of go fishing.”
He is fishing for cells, which ideally will search out the ITO electrodes and stick to them. Then, he will use highly sensitive electronics to detect the amount of electrical current. Lines spiking on the computer indicate that the cell is squirting electrons into the electrode. And his research has shown that about a million electrons a second cross from the bacteria and land on the electrode.
El-Naggar explains that Gross has taken his initial experiment a step further.
“The kinds of measurements I did myself in Berkeley in 2009 were non-biological measurements,” El-Naggar said. “Crude tools to assess the conductivity of the wires. Now we’re at a stage where we’re grabbing individual cells live and measuring respiration.”
Suddenly, one of those live cells appears – a tiny blob darting through across the screen.
“Oh, there’s a little guy,” Gross says. “Where’d you go, buddy?”
And just as suddenly, the image turns black.
“Uh oh.” His face becomes serious. “It looks like a little atomic bomb went off on my electrode.”
The electrode, he explained, had gotten fried by the machine’s laser. It’s two full days of work — gone.
“So many dead cells,” he said. “It happened so fast.”
Put simply, he explained, the laser was too strong and the electrodes too thick. It’s the kind of troubleshooting that happens all the time.
“A large number of questions we’re answering every day are like, ‘What’s the best way to glue these things together?’ or whatever mundane thing we’re doing, he says. “Once you have that figured out, you use it to answer a deeper question.
Among those deeper questions: What kind of electrode material is best? Should the electrode be rough or smooth? What are the best ways to grow the bacteria, so they’re most likely to stick to the electrode? How strong should the laser be to avoid these tiny massacres?
Failure, El-Naggar said, comes with the territory: “I have never myself designed and built an experiment that worked the second or third time. The key thing is to have the right temperament and attitude about science. A lot of people would be incredibly frustrated to waste two days of work.”
A proud physics tradition
John Regan is a professor of environmental engineering at Penn State University. He researches how engineers can harness microbes to convert waste to electricity. El-Naggar’s work, he said, gives researchers insight into the basic mechanics of such systems and has implications for how different microbes interact.
“We want systems that work at a high rate and that have a stable performance, so are resilient to changes in waste strength or waste composition,” he said. “With Moh’s work, if we know better how microbes do this cellular electron transfer, we may be able to design or operate a system that gives the microbes we want a competitive advantage.”
El-Naggar says his goal is to be a cheerleader and protector of his students. He seeks funding, speaks at conferences and plans projects so that they have more time in the lab.
“I try to protect my students by keeping these things away from them, so they can focus on the exciting science, the research,” he said.
This is especially true considering the do-it-yourself philosophy of his lab, where students build most components of the experiments themselves.
“Every Ph.D. student is essentially trying to do something original, so you can’t just buy a kit at the store,” Gross said. “You engineer from spare parts whatever you need to get it done. We end up being our own artisans to a degree.”
That homegrown quality is a proud physics tradition, El-Naggar added.
“Chemists study chemicals. Biologists study what’s alive. Physics is more mercurial than that,” he says. “It’s about the way we do things. Physics is one field that isn’t defined by the subject of the study as much as by the approach of the study. And the way we do things doesn’t matter, as long as we have a very quantitative approach.”
In this case, each experiment, each question answered, brings the scientists closer to the basic physics of how organisms move electrons very long distances. But it goes beyond that.
On one hand, El-Naggar says he hopes the research will ultimately lend itself to new technologies: using microorganisms to clean wastewater, power fuel cells, or build better solar power materials, for example. But more importantly, he says, it’s about curiosity.
“We do this because we’re curious about the world,” El-Naggar said. “Here is one particular example of something microorganisms have been doing for billions of years, and we’re only learning how they do it now.”
And there’s a lot of power in that.
This piece was updated on June 23, 2014.