A Creature From an Alkaline Spring Could Improve Biofuel Processing

A visiting scientist at Berkeley Lab tests double-threat microorganisms that can tolerate alkali and break down cellulose.

The only truly practical biofuels will be those made from abundant feedstocks like switchgrass, wheat straw, and other woody plants, whose cell walls consist of lignocellulose. After pretreatment to remove or reduce the lignin, the sugary remains of cellulose and hemicellulose are fermented by microorganisms to yield the biofuel.

“Each additional step in the process adds to the cost,” says Michael Cohen, a professor of biology at Sonoma State University who has discovered bacteria that could, with a little help from bioengineers at the U.S. Department of Energy’s Lawrence Berkeley National Laboratory (Berkeley Lab), increase the efficiency and reduce the cost of biofuel processing. “The species of bacteria we’re testing may be able to combine two important steps into one.”

Cohen found the unique strain of bacteria, which can tolerate high alkalinity and degrade cellulose at the same time, in a strange and isolated part of California called The Cedars, located inland from Timber Cove in the state’s Outer Coast Range. The site’s deep canyons and rocky serpentine barrens, all but invisible from the area’s few public roads, create a biological island that is home to living things rarely seen elsewhere.

Although industrial pretreatments for reducing lignin range from steam explosions to acid baths (low pH), Cohen knew that some of the most effective technologies soak the feedstock not in acid but its opposite, highly alkaline liquid (high pH), which breaks up the woody matrix to release the digestible hydrocarbons, the sugars. Only then are the bugs – microbes – added to do the fermenting.

What drew Cohen to The Cedars were its spectacular travertine springs, formed of calcium carbonate from eroding serpentine rock. On the logarithmic pH scale the spring water reached a remarkable pH 11.9, compared to pure water’s pH 7. With help from botanist Roger Raiche, who has led the effort to preserve The Cedars as an ecological preserve, Cohen took samples of decaying plant material and the diverse community of microorganisms in the springs.

“It was a case where nature was mimicking an industrial process, soaking the woody material in a high pH solution and breaking down the cellulose with microorganisms,” says Cohen. The task was to identify and isolate which bugs were the cellulose degraders.

Out of the woods and onto the lab bench

As a participant in DOE’s Visiting Faculty Program at Berkeley Lab, which supports research by outside faculty through collaborations with Lab scientists, Cohen worked with expert microbiologists Tamas Torok, Hoi-Ying Holman, and other members of the Earth Sciences Division.

Torok searches for extremophiles, bugs that thrive in harsh environments such as intense heat or radiation, in places like the Chernobyl Exclusion Zone, the steppes of Central Asia, and the mud pots of the Kamchatka Peninsula. Holman, the director of the Berkeley Synchrotron Infrared Structural Biology (BSISB) program at the Lab’s Advanced Light Source, studies real-time chemical processes in living cells on the microscopic scale.

On samples of ground-up switchgrass Cohen cultured communities of microorganisms, both bacteria and fungi, from decaying plants in alkaline spring water from The Cedars. Once the cultures were established, they were maintained in water that imitated the chemical composition of the original spring water. Using synchrotron infrared spectromicroscopy at the BSISB, Cohen and his colleagues were able to isolate eight or nine active cellulose degraders. One stood out.

“It was a strain of Cellulomonas bacteria, the only strain we extracted under oxygen-depleted conditions,” says Cohen. The FA1 strain was either aerobic (thriving in air) or anaerobic (living without air) depending on its environment. It could survive without oxygen, but, unlike most anaerobes, oxygen would not poison it.

FA1’s response to highly alkaline conditions was similar. “Cellulomonas FA1 is not an extremophile,” Cohen says. “It doesn’t love high pH, and in fact it would prefer a more neutral environment – but it’s highly alkali tolerant.”

Promising as it is, Cellulomonas FA1 leaves much room for improvement. “Unlike yeast, bacteria have many fermentation pathways, so when it comes to making biofuel contenders like ethanol, FA1 probably can’t make much,” Cohen says. Of the Cellulomonas species that can excrete ethanol, most would die if the ethanol concentration exceeded one percent. Only a few can tolerate up to 12 percent.

Turning an organism with remarkable properties – the ability to withstand extreme alkaline conditions and a complete lack of oxygen while degrading cellulosic biomass – into an efficient producer of biofuel is a challenge for the metabolic engineer.

In fact, it’s a Billion-Ton Challenge, described in DOE’s strategic analyses of U.S. agriculture and forestry resources for providing a billion tons of sustainable biomass annually, with the aim of reducing the country’s petroleum consumption by 30 percent. With the biomass available, the challenge becomes one of industrial technique.

“That challenge is beyond our capacities at Sonoma State University, but it could be done at Berkeley Lab – I’m already at work on the proposal,” says Cohen. “Cellulomonas FA1 is the best starter organism I could think of.”

Synchrotron Infrared Unveils a Mysterious Microbial Community

Berkeley Lab scientists join an international collaboration to understand how archaea and bacteria work together deep in a cold sulfur spring

Paul Preuss

Read more: Synchrotron Infrared Unveils a Mysterious Microbial Community

Waves of Berkeley Lab Responders Deploy Omics to Track Deepwater Horizon Oil Spill Cleanup Microbes

In the aftermath of the Deepwater Horizon oil spill in the Gulf of Mexico two years ago, various strategies were deployed to prevent 4.9 million barrels of light crude oil from fouling the waters and reaching the shores. A team of Lawrence Berkeley National Laboratory (Berkeley Lab) researchers found that nature also played a role in the dispersal process as marine microbial communities responded to the oil plume that made its way from the wellhead at a depth of 5,000 feet to the surface of the water.

“There was oil on the surface and oil below, but no oil in between,” said microbial ecologist Terry Hazen, former head of the Ecology Department at Berkeley Lab and now holder of the University of Tennessee-Oak Ridge National Laboratory Governor’s Chair. In a report published a few months after the oil spill, Hazen’s team reported that the microbes in the water were partly responsible for the oil’s disappearance. While several studies have since confirmed that various microbes played a role in the dispersal of the oil in the Gulf, understanding the composition of the microbial community and the roles of its members has not fully achieved until now.

Photo: A surface slick in the Gulf of Mexico, taken ~1.5 km from the
Deepwater Horizon wellhead (Olivia Mason, LBNL).

To learn more about the microbial community’s response to the oil spill, researchers led by Berkeley Lab senior scientist Janet Jansson availed themselves of the expertise and resources at two of the Lab’s national user facilities, the U.S. Department of Energy Joint Genome Institute (DOE JGI) and the Advanced Light Source (ALS). The work done by the Lab’s disaster response team demonstrated Ernest Lawrence’s pioneering vision of team science. The findings, published in two separate articles, track a series of microbial species dominating the community in the waters at various time points to remove different fractions of the oil.

As reported in an article published online June 21, 2012 in The ISME Journal, the team describes using a combination of genomics techniques to study the way the microbes responded to the influx of oil. They focused on the community’s expressed functional information or metatranscriptome. In addition, they isolated single cells to identify the predominant microbial members in the deep ocean oil plume. Using the latter technique, they were able to assemble a draft genome of what they say is the first deep-sea, oil-eating bacterium from a single cell.

Read more: Waves of Berkeley Lab Responders Deploy Omics to Track Deepwater Horizon Oil Spill Cleanup Microbes

Hoi-Ying Holman and Gregory Enns were interviewed by local radio station KWMR for their Hot Tech / Cool Science show on May 17, 2012.  They discussed the recent cellular studies observing protein phosphorylation.  You can listen to the broadcast here:

Berkeley Lab scientists demonstrate the promise of synchrotron infrared spectroscopy of living cells for medical applications

Berkeley Lab scientists observed phosphorylation in living PC12 cells stimulated by nerve growth factor as they differentiated and sent out neuron-like neurites. The researchers imaged individual cells and simultaneously obtained absorption spectra using synchrotron radiation from the Advanced Light Source. Cells not stimulated with nerve growth factor did not differentiate and showed different infrared absorption spectra.

Knowing how a living cell works means knowing how the chemistry inside the cell changes as the functions of the cell change. Protein phosphorylation, for example, controls everything from cell proliferation to differentiation to metabolism to signaling, and even programmed cell death (apoptosis), in cells from bacteria to humans. It’s a chemical process that has long been intensively studied, not least in hopes of treating or eliminating a wide range of diseases. But until now the close-up view – watching phosphorylation work at the molecular level as individual cells change over time – has been impossible without damaging the cells or interfering with the very processes that are being examined.

“To look into phosphorylation, researchers have labeled specific phosphorylated proteins with antibodies that carry fluorescent dyes,” says Hoi-Ying Holman of the U.S. Department of Energy’s Lawrence Berkeley National Laboratory (Berkeley Lab). “That gives you a great image, but you have to know exactly what to label before you can even begin.”

Holman and her coworkers worked with colleagues from the San Diego and Berkeley campuses of the University of California to develop a new technique for monitoring protein phosphorylation inside single living cells, tracking them over a week’s time as they underwent a series of major changes.

“Now we can follow cellular chemical changes without preconceived notions of what they might be,” says Holman, a pioneer in infrared (IR) studies of living cells who is director of the Berkeley Synchrotron Infrared Structural Biology program at Berkeley Lab’s Advanced Light Source (ALS) and head of the Chemical Ecology Research group in the Earth Sciences Division . “We’ve monitored unlabeled living cells by studying the nonperturbing absorption of a wide spectrum of bright synchrotron infrared radiation from the ALS.”

The researchers report their results in the American Chemical Society journal Analytical Chemistry.

Read more: Molecular Spectroscopy Tracks Living Mammalian Cells in Real Time as They Differentiate