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The Rise of the Biological Fuel

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What do E. coli, grass, pond scum, and wood fungus all have in common? They could all have a hand in saving the planet. No, that is not an obscure science joke — it is the culmination of research from all over the world on the best ways to replace our stores of slowly dwindling fossil fuels with sustainable, renewable biofuels. According to the US Department of Energy, nearly 85 percent of all energy used in the United States comes from fossil fuels — coal, oil, and natural gas — and there's a big possibility that that number will increase during the next two decades. The US government, the European Union, and corporations such as ExxonMobil and Chevron are teaming up with scientists to come up with a viable solution — or set of solutions — to the problem.

Some researchers are exploring ways to more efficiently break down the crops we already grow as sources of biofuel to get as much energy as possible out of them. Others say the answer lies in finding new sources of energy that do not require the planting of millions of acres of feedstocks. Yet others are trying to sequence and engineer organisms found in nature to synthesize processes similar to how those organisms naturally create energy from sunlight, bacteria, or carbon dioxide. What these researchers all have in common is that they tend to agree that biofuels are the way of the future, and that it's going to take a combination of techniques, products, and natural resources to get the job done.

The advent of biomethane

Not long ago, rumblings of apprehension were brewing about the way biofuels were being produced in the United States. In January 2008, it was estimated that about 20 percent of the 93 million acres of corn -planted in the US in 2007 was being used to produce ethanol. Prices of corn, beef, poultry, milk, and pork were on the rise, and no one was seeing any benefit or any measurably decreased use of oil and natural gas. Not only was food being used — some said needlessly — to make fuel, but the process for doing it took too much energy.

Around that same time, in Europe, farmers were being asked to grow rapeseed, also called canola, for the purposes of making biodiesel, but the same problems existed — too much energy in, not enough out. Jerry Murphy, the lead investigator at University College Cork's Sustainable Energy Research Group in Ireland, and his team set out to do life cycle analyses of several different sources of biofuels to find the most efficient and sustainable, starting with rapeseed.

"We were quite apprehensive about [rapeseed] because when you analyze it, the energy return in a unit of land is very low," Murphy says. "If we converted one percent of agricultural land to rapeseed, we got one percent of transport fuel. It seemed a ridiculous concept to be converting land to [grow] a fuel that would be giving you such a low return per hectare." Murphy's group also studied the efficiency of using tropical fuel sources such as palm oil from Thailand. Though the energy return from palm oil is high — even factoring in the energy it would take to transport the fuel to Europe — the impact on the area's ecosystem was also high. Palm oil was banned in the Netherlands because of its ecological impact. "We just felt that biofuels were losing the war," Murphy says. "There was a really strong backlash against them."
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Murphy and his colleagues started looking at sources of renewable natural gas instead. They visited an abattoir in Sweden where they noticed that part of the waste the facility produced was fermented belly grass — about 100 kilograms per cow. That started them thinking that the grass itself could be turned, through a very simple process, into biomethane. The group visited digesters in Europe that were breaking down grass — a process that takes around 60 days — but when running experiments of their own, and keeping in mind what they had seen at the abattoir, they added a little rumen to the grass, sealed everything in a container, and found that after two days, they had the same rate of destruction of the biodegradable material.

"If you put grass into a tank without oxygen, you get an awful lot of gas," Murphy says. "We found that the energy from a unit of land in Ireland was five times better for grass than rapeseed. You don't import anything, you don't plow the land, and the system is very simple — you put grass into a tank, and you heat it to 38 degrees Celsius, and gas comes [out]." The simplicity of the process is especially important considering the European Union's Renewable Energy Directive, which states that by 2018, if a fuel is to be considered a biofuel, it will have to produce at least 60 percent more energy than the fossil fuel it is replacing. The return from rapeseed biodiesel is only about 35 percent, Murphy says, while wheat ethanol gives a return of about 25 percent. Biomethane, on the other hand, gives 85 percent more energy than the fuel it replaces, natural gas. Europeans also do not consider something a biofuel if the land has to be plowed to create it. The energy demand to make ethanol from wheat or corn is very high, and to create 100 million gallons of it, you need to use 500 million gallons of water, Murphy adds. With grass, there is no such requirement.

"We're already injecting the bio-methane into the gas grid," Murphy says. Once the grass is fermented, all that is needed is to take out the carbon dioxide, and what is left is about 98 percent methane. In Ireland, and all over Europe, this biomethane is being used to provide electricity and to power buses and cars that run on natural gas.

Next step: algae

The next step, Murphy says, is the widespread use of algae as an alternative to petroleum. "Algae is going to be the holy grail," he says.

For many researchers like Murphy, algal biofuels are the next big step in biofuel innovation. Among the thousands of independent researchers and companies racing to put themselves on the map for developing the most efficient and highest-producing algal biofuel are none other than Craig Venter and ExxonMobil, who have teamed up in a $600 million project to find which strains of algae make the best fuel.

Mark Hildebrand, a researcher at the Scripps Institution of Ocean-ography, is also one of the many researchers trying to find the best way to turn algae into a fuel powerhouse. Hildebrand's work is mainly focused on the unicellular alga diatom, which, he says, is responsible for about one-fifth of the total carbon fixation on the planet, is very abundant, and is very good at accumulating neutral lipids — biofuel precursors — that can be converted to a biodiesel. "They're very successful in the ecological sense," Hildebrand says. "They accumulate the lipids very well in the lab, in very high amounts and very quickly."
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The problem with algae — a problem that many researchers are currently wrestling with — is trying to scale it into large-volume production. The yield researchers get in the lab does not always translate to large-scale outdoor production. Hildebrand and his team are currently trying to understand what factors influence that process, even trying to genetically modify the organisms to produce lipids more consistently, or selecting for mutant strains that perform better outside the lab. "We're really looking at metabolic changes going on in the cell, and we think that's partly triggered by gene expression," Hildebrand says. "We see kind of a dual induction when the cells start to accumulate their lipids. There's a certain rate of accumulation over time and then there's some transition — and we don't know what it is yet — where they'll double the rate after they reach a certain stage."

The researchers are reasonably certain that the first stage of this dual induction process is a change in the diatom's gene expression and in its metabolic pathway fluxes, but after that, there is an effect on the cell where large-scale changes happen in the flux of carbon in the cell. It is this second stage that Hildebrand's team is carefully studying. "When the cells are normally growing, they make carbohydrates for growth and they don't accumulate lipids. They do that only when they stop growing," he says. "And so what we're hoping to do is understand what's causing that change in the carbon flux from carbohydrates to lipids and if we can understand that, the idea would be to metabolically engineer the cell so they can constitutively make lipids. Instead of it being a two-stage process where you grow them and then produce lipids, you could cut that down to one stage where they grow and produce lipids at the same time."

Despite this uncertainty, there is "no question" that algae is the most promising avenue to a sustainable biofuel, Hildebrand says. The algae only need sunlight and carbon dioxide to produce the lipids necessary for conversion to biofuels. Because they use atmospheric carbon dioxide, they do not introduce new carbon dioxide and they do not have to waste energy fighting gravity to grow or build superstructures like root systems to survive. Growing the organisms would not require using up land for crops or potable water, Hildebrand says. They can be grown in areas of high sunlight — deserts, for example — where crops cannot be grown, in ponds of saline or wastewater.

The trick is to make the lipid-production process more consistent to improve the economics of algal biofuels, Hildebrand adds. "We're really close to seeing a biofuel made with algae. The issue then becomes generating infrastructure to scale this up — growing algae in large outdoor ponds or enclosed photoreactors. That technology needs to be developed better," he says. "It's an engineering problem, and I'm sure it will be solved. But to me, that's where the big limitation is right now."

In the works

The production of an actual biofuel from grass and algae, though the most advanced strategies at this stage, are not the only sources of inspiration for researchers looking to abate our energy woes. Some researchers — and biotech companies — prefer to go another route: finding the most energy-efficient ways to turn feedstocks we already use into bioethanol.
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At the US Department of Energy's Joint Genome Institute, researchers like Igor Grigoriev are hard at work studying how various species of wood fungi enzymatically break down biomass. Grigoriev and his team have just finished sequencing the white rot fungus Schizophyllum commune in an effort to learn how it converts cellulose into simple sugars and then into ethanol, with an eye toward developing an equivalent industrial process. S. commune is the second white rot fungus and the third wood degrader that JGI has sequenced, starting with Phanerochaete chrysosporium in 2004 and the brown rot fungus Postia placenta last year.

"The goal of the fungal program is to scale up sequencing and analysis of fungi to explore phylogenetic and ecological diversity of fungi," Grigoriev says. "The idea is to sample the diversity and learn from it. We're just touching the surface of all this knowledge encrypted in fungal genomes." The brown fungal rot, for example, was found to use small molecules that break down cell walls and decompose wood into simple sugars. But the white rot uses a different mechanism to break down cellulose and lignin with completely different enzymes, and by invading the xylem tissue. "We need to sequence much more to get an idea of variation, and what of that variation could be used [in an industrial process]," Grigoriev says.

The researchers are also starting to experiment with genetic manipulation of the rot, which is a mushroom, to enhance the volume of enzymes being produced. Fungal gene-knockdown work is going slowly — some knockdowns kill the mushrooms while others cause them to proliferate. There is also ongoing research to see if inserting genes and tweaking their expression levels has any effect on protein production in the fungi. So, Grigoriev adds, there is plenty of work yet to be done.

He and his team are collaborating with biotech company Novozymes to optimize and commercialize these enzyme mixes for the production of biofuels. In fact, Novozymes has been collaborating with JGI for the past eight years on various projects that have one thing in common — advancing the efficiency and usability of biofuels. Novozymes doesn't directly make or sell biofuels, says company director Randy Berka, but the company's enzyme mixtures are its way of contributing to the biofuel boom.

"We recognize that there's an incredible diversity of organisms on the planet that deploy an assortment of strategies and enzymatic machinery to accomplish the task of recycling biomass carbon," Berka says. "We're going to need a lot of feedstocks if we're going to replace current petroleum-based fuels. There's plenty of room for a lot of different technologies." But the time and money Novozymes has put into its collaboration with JGI has been worth it for the company. "Liquid fuel from biomass represents one of the first steps of a bio-based society, and that's where Novozymes wants to go," he adds.
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JGI public affairs manager David Gilbert says the work Grigoriev's group is doing with fungi is a complementary strategy to the work other JGI researchers are doing with enzymes sourced from termite guts and cow rumen that can break down grasses and other feedstocks. "The DOE is looking into several strategies, products, and sources of bio-fuels," he says.

There is also work underway to find methods of utilizing fatty acid-rich feedstocks, which are not currently used in biofuels. At Rice University, Ramon Gonzalez's research group has been working for the past couple of years to find a way to use E. coli as a vehicle for the fermentation of fatty acid feedstocks into bio-ethanol and biobutanol. These feedstocks are abundant, have an efficient metabolism, and have higher yields and higher titers of biofuels than feedstocks currently in use. Clementina Dellomonaco, a graduate student in Gonzalez's group, says that fatty acid feedstocks are used for chemical production to biodiesel but that there is not currently any biological process in place that can convert the fatty acids into fuels. Since fatty acids are metabolized only under respiratory conditions and not through fermentation, the researchers have had to engineer E. coli to create a "respirofermentative metabolic mode" that enables the synthesis of fuels from fatty acids, Dellomonaco says. "The metabolically engineered bug is able to eat up the fatty acids and convert them into high-value products," she adds. The engineered bug has had all competing pathways deleted so only the pathway that produces the desired bioethanol is left. In the case of biobutanol, the researchers introduced a synthesized pathway from the Clostridia bacterium into the E. coli to produce a non-native product.

The sources, methods, technologies, and innovations in the world of biofuels development are many. Some are ready for wider use now, while others still have several years of testing and optimization to go through. But researchers hope that soon a variety of products and processes will exist to make the world's dependence on fossil fuels less and less, until it is eventually gone.

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