Breaking Down the Wall

Every Labor Day for several years, long distance runner Rod Mackie would compete in a road race that went past a canning factory garbage dump just outside Hoopeston, Il. As a microbiologist at the University of Illinois at Urbana-Champaign, he couldn’t help noticing a smelly, bubbling, porridge-like leachate that was oozing up from the ground.

Intrigued, he finally stopped after a race in 1993 to take a sample of what turned out to contain a versatile bacterium later named Caldanaerobius polysaccharolyticus – a heat-loving microbe that may be valuable in making enzymes for biofuel production.

Professor Mackie, a gut microbiologist, wasn’t thinking about biofuel when he took the sample. He was looking for what he calls a “hot Beano” –  heat-resistant enzymes to break down certain indigestible components of gas-producing foods like beans.

Dr. Rod Mackie, University of Illinois at Urbana-Champaign

But his college also happened to have a fund for plant cell wall degradation and biofuel research. “As we were working with this particular bug, we knew it had a vast array of enzyme activities,” he said. That started them thinking about what was in that bacterium’s genome. It turned out to have a lot of enzymes capable of breaking down insoluble polysaccharides in plant cell walls—the first step in making second-generation biofuel.

A mighty fortress is a plant

That serendipitous bit of bioprospecting (see "Hot Spots," left) highlights the new ways researchers are mining nature for tools in the quest for a commercially viable advanced biofuel – a sustainable alternative to our dwindling fossil fuels.

There’s a treasure trove of bioenergy locked up in lignocellulose – the structural part of plant leaves, roots and stems. For most of human history, combustion of lignocellulose in the form of wood was the main source of energy. Approximately 10 percent of all human energy use today is still derived by combustion of lignocellulose, yet it is estimated only about 2 percent of the earth’s annual supply is utilized by humans.

The problem in using lignocellulose for biofuels is getting at it. That’s because plants have evolved fortress-like cell walls over 450 million years, both to hold themselves upright and move water and protect themselves from disease and predators.   

These cell walls are composed of lignocellulose, made up of three types of polymers -- cellulose, hemicellulose, and lignin – that are knitted into a complex polymer matrix surrounding the plant cells. The cell walls make each cell rigid, allowing plants to grow upright (see illustration, below).  

Components of the plant cell wall

The most abundant component in plant cell walls is cellulose, which comprises about half of the overall mass. At the molecular scale, cellulose microfibrils resemble long ropes composed of chains of glucose molecules (polysaccharides) that wrap around the plant cells to make them stiff.  Cellulose is familiar to most people because it is the main component of cotton and paper and is the most abundant organic material on the surface of the earth.  Hemicellulose, which is less well known, is composed of other sugars such as xylose and arabinose and coats the cellulose microfibrils to prevent them from sticking to each other.  (In fact, paper is made by separating hemicellulose and lignin from cellulose so that the cellulose molecules adhere to each other without the need for any glue.)  Then there is lignin, which is composed of cross-linked phenolic compounds that stubbornly resist breakdown.  It accounts for about one-fifth of lignocellulose. Not only is lignin a major component of coal (lignite) and soil, its molecular structure is somewhat similar to the PET plastic commonly used in water bottles.  Like plastics, lignin is a very durable material that is not readily decomposed by biological processes.

Because it is so difficult to decompose, lignin that is produced as a byproduct of advanced biofuel production is usually burned to provide heat and electricity for the overall biofuel production process.

To make ethanol and other liquid fuels from lignocellulose, the most common approach is to use enzymes to break up the polysaccharides into free sugars and then use microorganisms to ferment the sugars to ethanol or other fuels (such as butanol, another type of alcohol).

Most of the methods in these steps of biofuel production come directly from the brewing industry, in which the sugars in grains or grapes are fermented to the alcohol in beer and wine. In fact, so-called "first generation" biofuels from corn or sugarcane are essentially 200 proof bourbon and rum.

Unlike first-generation biofuels, which are made from sucrose or easily degraded starch, the main challenge in making liquid fuels from lignocellulose is in breaking up the polysaccharides that comprise cellulose and hemicellulose into free sugars.  At present, plant biomass is usually “pretreated” with an acid or base that partially disrupts the plant cell wall structure so that enzymes can penetrate the biomass and depolymerize the polysaccharides to unlock the sugars – a process some scientists have dubbed “deconstruction.”

It’s a time-consuming and difficult process, thwarting nature’s barricade.  Depolymerization is the biggest challenge and the most expensive step in creating biofuel from plants, accounting for more than 50 percent of the costs, says Blake Simmons, Ph.D, Chief Science and Technology Officer and Vice-President of Deconstruction at the Joint BioEnergy Institute in Emeryville, Calif.

Looking for sustainable solutions

Dr. Isaac Caan, Professor of Microbiologiy and Animal Sciences, University of Illinois

At first glance, it seems strange that it is difficult to deconstruct the plant cell wall because it is a process that happens naturally on a massive scale. The earth’s ecosystems constantly produce plant biomass that eventually dies and is decomposed by a variety of organisms. Termites, cows and other ruminants, and other creatures consume some of the biomass, though they all rely on a consortium of microorganisms in their guts to break down the polysaccharides to sugars that they can then metabolize. And recently, EBI deputy director Isaac Caan and his colleagues at the University of Illinois discovered that some of the best microbial candidates for this process may actually reside in the human lower intestine.

Other biomass is decomposed by free-living microorganisms such as filamentous fungi, which depolymerize the lignocellulose and consume the sugars and, in some cases, the lignin. Small wonder that one of the fundamental ideas underlying much of the research on advanced biofuels is deceptively simple: by understanding the processes used in nature to decompose biomass, we may be able to adapt some aspects of those processes in our goals of producing liquid fuels and chemicals.

“We’ve created entire industries to try to do something that bacteria [and fungi] have been doing naturally for millions of years,” says Paul Gilna, Ph.D,  director of the Department of Energy’s Bioenergy Science Center in Oak Ridge, Tenn.

Dr. Timo Schuerg, University of California at Berkeley

Although scientists are exploring methods for decomposing biomass with high temperature processes or strong acids, the idea of adapting natural bioconversion processes is attractive because they are nonpolluting and renewable.

“We can do amazing things with technology, but very often it’s highly energy expensive,” said Timo Schuerg, Ph.D, a postdoctoral researcher who is studying the secrets of plant deconstruction by fungi at the University of California, Berkeley.

“We need to look deep into nature and try to understand its sustainable solutions," Schuerg says. "We need to ask, how is nature doing it? That is the only way for us to achieve sustainability.”

Nature’s own deconstructors: Microbes

The special toolkits of enzymes produced by certain microbes hold out promise for renewable ways to unlock sugars from biomass. Mackie’s garbage bug is one of many such specialized microbes discovered around the globe, from hot springs in Nevada, Iceland, and  Yellowstone Park to backyard compost piles. Just as miners once prospected for gold in streams and rivers, scientists go on bioprospecting expeditions to look for these microbes, part of a class known as extremophiles, or “lovers of extremes.” (See sidebars)

Of course, no enzyme can do the job alone. Although plant cells walls usually have two main types of polysaccharides, the sugars that comprise the polymers are linked together in many different ways. Since each enzyme usually breaks just one type of sugar-sugar chemical bond, many different enzymes are needed to unlock the sugars in lignocellulose.

Extremophiles that may be useful in bioenergy have been found in the Valley of Geysers in Kamchatka, Russia (Credit: Dreik39/Dreamstime)

And when it comes to biofuel production, certain types of enzymes are more equal than others. Heat-loving (thermophilic) microbes and their enzymes, active at temperatures  as high as 200 ºF that kill just about anything else, are especially well-suited for a process that often involves extreme heat during pretreatment.  

Because different microorganisms have adapted to decomposing different types of biomass in everything from acid lakes to boiling geysers, they produce enzymes with many different properties. Scientists are exploring the properties of enzymes produced by microbes in different ecological niches in the hope of finding the enzymes that can withstand the extreme heat, acidity, and other harsh conditions in the industrial process to create biofuel and other products.  Among the most coveted: Enzymes that are durable, heat-tolerant, have an acceptable Ph range, and are not inhibited by the other biocompounds in the process.

According to Douglas Clark, Ph.D, Chair of Chemical and Biomolecular Engineering at UC Berkeley and a principal investigator at the Energy Biosciences Institute (EBI), harnessing these microbes and their enzymes could allow biofuel producers to reduce the energy required to cool biofuel reactors. And because thermophilic enzymes are usually more stable -- and chemical reactions are accelerated by temperature -- it may take fewer of them to get the job done.

“If we can use fewer enzymes, that would be a major breakthrough because it would reduce the cost,” Clark says, noting that enzymes are expensive.

Dr. Chris Somerville with senior analysis fellow Heather Youngs, Ph.D., University of California at Berkeley

Scientists are searching for promising candidates with the tools of advanced imaging and DNA sequencing, used to study how microbes break down cellulose and hemicellulose into simple sugars. In one EBI project, using high throughput DNA sequencing, Mackie, Clark and other EBI researchers have discovered more than 27,000 carbohydrate-degrading enzymes in the rumen fermentation compartment of the cow stomach. 

Potentially useful enzymes have also been found in the guts of termites and tiny wood-eating marine pests called gribbles.

“We are awash in biodiversity,” notes Chris Somerville, director of the EBI. “Finding the best enzymes among so many candidates is an overwhelming task.”

A future with fungi

Bacteria are not the only microbes eating biomass. Fungi have evolved over millions of years, to become one of nature’s best – and most prolific—plant cell wall deconstructors.

“Fungi are the real experts in breaking down cellulose,” said Schuerg. If we didn’t have fungi, he adds, “we would have a big problem with cellulose waste”—all the leftovers from trees, grass, and harvests would be overwhelming.

At the CBS-KNAW Fungal Diversity Centre in the Netherlands, researchers are genetically barcoding the complete collection of more than 75,000 fungal strains, which are publicly available for use in bioenergy and other research areas.

“People keep looking for a super cocktail, usually from one fungal strain, that will do all the work,” says CBS scientist Dr. Ronald de Vries. “But the enzyme mixture that fungi produce tends to change over timeas they degrade biomass, while in contrast commercial cocktails are static in their composition. To achieve the same efficiency as the fungi, the cocktail will probably need to be spiked with specific enzymes during the saccharification process.”

“Also, a fungus in nature has no aim to fully degrade biomass,” de Vries points out.  “What it wants to do is find a food source, propagate itself, and try to stay alive. There is not a single fungus in the world that is truly dedicated to fully degrading biomass. But that’s what we want it to do. So if we can identify the complex strategy by which it decides what to break down, and be able to combine the strategies of several fungi in our commercial process,  we’ll be closer to that goal.”

An ocean away at UC Berkeley, Schuerg is working on a model filamentous fungus, Neurospora crassa, a fluffy orange fungus often seen growing on trees after a fire. Valued by scientists for the ease of its genetics, biochemistry and molecular biology, it’s seen as potential game-changer for bioenergy. Schuerg is investigating how it makes cellulases (enzymes that can chop up cellulose). “For biofuel we need a huge amount of enzymes,” he says. “They are still pretty costly, so it’s a bottleneck in biofuel production.”  

Dr. Louise Glass, Chair of Plant and Microbial Biology, UC Berkeley

Fungal geneticist Louise Glass, Ph.D., Chair of Plant and Microbial Biology at U.C. Berkeley, has acclaimed N. crassa’s virtues in a lecture called “Neurospora crassa: Portrait of a Fabulous Fungus.”  (Indeed, several postdocs spoke of the fungus with deep affection, with one comparing it to a beloved “lab pet.”) Along with U.C. Berkeley Professor Jamie Cate, Ph.D., of the molecular and cell biology department, she and other researchers at the EBI have taken genes from the grass-eating fungi and inserted them into yeast, creating strains that are able to use sugars that are normally not metabolized.  Another goal is to engineer the fungus to overproduce target cellulase enzymes at will. She has explained that what the researchers hope to produce in N. crassa is a blueprint for making inexpensive designer cocktails of plant cell wall-degrading enzymes. 

Today’s technology not only relies on the production of large amounts of enzymes - enzyme cocktails even need to be tailored to the many kinds of feedstock, which are “vastly different” in different parts of the world, said J. Philipp Benz, a post-doctoral researcher at U.C. Berkeley. In fact, “the composition of lignocellulose is very different even in different parts of one plant,” he said. “The enzymes will have to work with similar efficiency on all of these.”

Breeding better raw material 

U.C. Berkeley plant and microbial biologist Markus Pauly, Ph.D, is taking another approach: helping nature create plants that are better suited for biofuel production through spontaneous mutagenesis or targeted genetic alteration.

Plant cell walls have had to become extremely good at resisting breakdown because  “plants can’t run,” as Pauley puts it.

(“Of course, that hasn’t stopped them from surviving for millennia,” he adds.)

Because the lignin in cell walls is a barrier to releasing sugars, one way to improve plant composition for biofuels is to reduce the amount of lignin that plants make, “but then the plant lies flat on the floor,” says Pauly, who is a principal investigator at EBI.  Recently, he said, researchers have been able to program plants to make modified lignins that are chemically easier to break, but still keep plants upright.

Dr. Markus Pauly, UC Berkeley

Pauly’s approach is to make sugars more accessible, or to select for mutant plants that accumulate more sugar or polysaccharides. Working with corn, Pauly and collaborators randomly mutated seeds, screened the resulting plants for high levels of polysaccharides, and identified a new corn plant variety called Candyleaf 1 (or CAL1, for the University of California). The mutation inactivated a plant enzyme that naturally degrades hemicellulosic glucan, a type of polysaccharide found in leaves and stems. They ended up with a corn plant which yielded 30 percent more sugar after conversion of its lignocellulose. 

Corn grain has long been used to make biofuels, as it can easily be converted to ethanol. But using grain for fuel is problematic and controversial. The new variety created by Pauly and his lab at EBI presents a win-win: only the corn stover, or waste leaves and stalk, is processed to fuel, while the kernels can be used as a food crop. Because CAL1 is a non-transgenic, induced mutation, similar to those occurring in nature, there is no problem with genetically modified food controversy or regulation. The process could be used with other food crop residues from rice, wheat and so on, he said, adding, “You take and use the kernels for food and take the leftovers for fuel.”

Breaking down the research walls

Although small amounts of lignocellulosic ethanol are now being produced, there is a general sense in the biofuels community that additional innovation is required in order to achieve the kind of efficiency and profitability that will stimulate expansion of lignocellulosic fuels from about twenty million gallons today to more than a billion gallons a year.

Dr. Blake Simmons (right)  of the JBEI in the lab; credit:Sandia Labs

One of the hurdles is the huge variety of plants and plant structures scientists would like to use – all of which could need specialized deconstruction and extraction techniques. Simmons says the goal is “an omnivorous pretreatment technology” that can handle any range of feedstocks.

Startling progress is being made through cooperation among the many disciplines involved. Historically, as science progresses, it becomes more specialized and compartmentalized, but times are changing.

“We’re starting see these walls tumble,” said Simmons. The synergy of sciences and specialties at many research and academic institutions has been breaking down departmental barriers between chemistry, biology, botany, economics, engineering, waste management and animal science, to name a few.

The type of cross-disciplinary research practiced at JBEI and the EBI  “is a fundamental mind shift, and one of the greatest payoffs …is to bring together individuals from a wide range of backgrounds and disciplines to work on a common mission,” said Simmons. “The search for renewable and sustainable biofuel is calling up a new generation of renaissance researchers who will have an impact in far reaching fields.”

 

Chris Woolston, M.S., Timo Schuerg, Ph.D, and J. Philipp Benz, Ph.D., contributed additional reporting to this article.

 

Next issue: Other ways to break down biomass

 

 

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