Plant Matter Prowess

Rooting out problems in biofuel production

Plant matter is widely thought
of as a promising alternative
fuel source to petroleum. Of course, the key to unlocking lignocellulose—the main building block of plant cell walls and the most plentiful organic material
on the planet—is to break down its hearty matrix of cellulose, hemicellulose, and lignin among others, and to do it as simply as possible.

Turning plants into ethanol involves three steps: pretreatment, enzymatic hydrolysis, and fermentation.
But getting good yields is a challenge. Attempts at genetic engineering to make trees more digestible have led to less viable plants. Reaction times during fermentation are inefficient, in part because yeast used in fermentation doesn’t metabolize all of a plant’s sugars. Although producing the new and promising biofuel 2,5-dimethylfuran (DMF) involves fewer steps than ethanol production, the reaction can still take hours or days. And after researchers perfect conversion reactions...

The Scientist delved into these challenges, talking to four different groups that are improving some aspect of plant biomass conversion into biofuels. Here’s what we found.

Sweet Yeast

Project: Fermenting plant sugars for conversion into biofuels

User: Huimin Zhao, professor of chemical and biomolecular engineering, University of Illinois at Urbana-Champaign

Problem: Yeast species Saccharomyces cerevisiae is thought to be a promising organism for ethanol production from plant biomass. Unfortunately, the organism does not process five-carbon (pentose) sugars, which can make up as much as 30% of biomass. That’s partly because this yeast strain does not efficiently take up pentose: the molecule can only enter yeast cells through six-carbon (hexose) transporters with two orders of magnitude lower affinity. Zhao and his colleagues needed a way to increase pentose uptake in yeast strains so that it too could be used along with hexose sugars such as glucose and fructose.

Solution: Zhao’s group cloned and characterized three pentose sugar–specific transporters—one arabinose and two different xylose transporters—from various pentose-using species such as fungi. They expressed these transporters in S. cerevisiae. In preliminary experiments using radiolabeled pentose sugars, the engineered yeast did increase uptake of pentose sugar compared to uptake in unaltered yeast. In some cases, the engineered yeast produced a greater concentration of ethanol during fermentation. However, the fermentation results aren’t yet reproducible, Zhao says.

Next steps: The group is tweaking pentose transporters to work more efficiently in yeast. “The big challenge right now is that [pentose] sugar utilization is not as efficient as the glucose,” Zhao says. Glucose can be used in a day, whereas pentose sugars take longer than 2 days, he adds. One way to speed the process will be to engineer a pathway of enzymes so they can metabolize sugars quickly once they’re inside the yeast cells.

Big idea: Researchers hoping to improve pentose fermentation can discover new pentose-specific transporters by searching existing gene databases like GenBank using Zhao’s pentose-specific transporters as probe sequences (which he will describe in an upcoming paper). To confirm the functionality of these new transporters, he genetically expresses them in a yeast strain with most of its hexose transporters removed and then does a simple sugar uptake assay, Zhao says.

Poplar Engineering

Project: Breaking down plant biomass by tackling the root of the problem: the plant itself

User: Ming Tien, professor of biochemistry, in collaboration with John Carlson, Pennsylvania State University, University Park

Problem: Increasing the digestibility of plant biomass is one main way to improve yield from alternative energy sources. In particular, plants with less lignin in their cell walls are easier to break down and convert to fuel products. But genetic manipulations that decrease lignin content interfere with other plant functions. For example, lignin-deficient plants can droop, or they can be more susceptible to pests, Tien says.

Solution: Tien works with poplar trees, a good potential source for fuel partly because they grow relatively fast and can be pruned for fuel production instead of completely cut. Instead of decreasing the amount of lignin in the trees, Tien and his colleagues modified the connections between lignin molecules with help from a gene they borrowed from parsley and introduced into poplar trees. The gene produces a protein called “glycine-rich protein” that is rich in tyrosine and that can insert itself between lignin molecules. The protein can be targeted and broken down using specific enzymes. In the first generation of plants, the group saw a greater yield of sugars released from the transgenic poplar compared to genetically normal trees.

“The difficult part now is going to be in the details—what proteins? What size? What kind of amino acids do we put in there besides tyrosine? How much should we express?” —Ming Tien

Next steps: Tien’s group is now growing their second and third generations of poplars with enhanced tyrosine. They are also looking for other proteins that crosslink well with the lignin, but that make plants easy to break down chemically. “The difficult part now is going to be in the details—what proteins? What size? What kind of amino acids do we put in there besides tyrosine? How much should we express?” says Tien. “We first want to make bigger improvements in yield,” he adds. “Then we’ll back off and worry about [tree] fitness.”

Big idea: Although this strategy is not widely used yet, Tien says that in theory, it’s applicable to any plant that can be genetically altered. Switchgrass, the front-runner among plant biofuels, is not yet among these. For newbies altering lignin, “there’s going to be pitfalls here as far as the fitness of the new plants,” Tien says. The development process will involve the trial and error of altering the type of plant, the genetic engineering, and even the environment in which the plant is grown, to boost survival. Plant geneticists are essential to the process, Tien adds.

Nuke It

Project: Converting cellulose within corn stalk, rice straw, and pine wood into 5-hydroxymethylfurfural (HMF), a chemical intermediate that is easy to convert into the potential biofuel DMF

User: Zongbao Zhao, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, China

Problem: In the past few years, several groups have found ways to directly convert the cellulose in plant matter into DMF, a process that involves fewer steps than that used in ethanol production. Many of these reactions involve conventional heating systems such as an oil bath, but it takes hours or days using these methods for the reaction to occur. Zhao and his group wanted to speed up the process to take on the order of minutes.

Solution: Last year, Zhao and his group reported a one-step process that converts lignocellulosic biomass into HMF. Their success was based on the facts that cellulose can be efficiently broken down by adding an ionic solution to it and that chromium chlorides (CrCl3) can help speed up HMF formation from the plant’s glucose. Using a commercial 1100-watt microwave equipped with magnetic stirring system, Zhao’s group blended an ionic liquid solvent, CrCl3, and purified cellulose starting material. They heated the mix for 3 minutes and extracted HMF with a yield of 62% (Tetrahedron Lett 50: 5403–5, 2009). “In a few seconds you actually heat the inner bulk of material to over 150 degrees, and the reaction is done,” Zhao says, adding that because microwave heating significantly reduces the reaction time, “degradation and other side reactions of HMF are limited.”

Next steps: Zhao and his colleagues are working on a way to recycle the solvent, to reduce costs. They’re also looking for better catalysts to replace CrCl3, which is toxic. “We hope to find an equally good or better catalyst that is more environmentally friendly,” Zhao says.

Big idea: Although other groups have been using lithium chloride as a catalyst, Zhao says it’s important to use CrCl3 or CrCl2. “Other metal halides, such as lithium chloride, iron chloride, and magnesium chloride, had much less efficiency in this transformation,” he says. This year, Zhao’s group tried the method using plant matter; corn stalk, rice straw, and pine wood produced HMF in a yield of up to 52% in 3 minutes (Bioresour Technol 101:1111–14, 2010).

Scale Up

Project: Scaling up a biomass–biofuel conversion from 500-mL flask to a 22-liter reactor

User: Dave Lane, postdoctoral researcher in the laboratory of Pieter Stroeve, University of California, Davis

Problem: To be able to take a specific biofuel production process to a commercial scale, researchers first have to show that it is possible to scale up the conversion reaction. Stroeve’s group was faced with the nuances of scaling up the process. For example, in their small, sealed set-up, the flask could resist pressure to contain 12 M hydrochloric acid at 100 degrees Celsius. In a larger reactor, which must be operated at atmospheric pressure for safety reasons, HCl would evaporate from the reaction. They needed a constant, known concentration of acid to get usable data about the reaction kinetics.

“We haven’t yet gotten the rates up to what they were in the [smaller] pressurized reactor, but we are closing in on that goal.” —Dave Lane

Solution: Stroeve and his collaborators cut the HCl concentration to 6 M, which effectively helped them keep the liquid at a constant boil, so that it did not change while being distilled. “When you boil 6 molar hydrochloric acid, the vapor has the same composition as the liquid, so it is sometimes called ‘constant boiling’ hydrochloric acid,” Lane says. “It was important for us to use constant boiling acid so that we could have as high a concentration as possible without boiling away the HCl gas.” The heating system is quite different in the large reactor—a single heating tape fastened to the large glass reactor, as opposed to the hot oil bath for the small flask.

Next step: Although it takes 2 hours to heat the reaction, with the lower concentration of HCl, the group is hoping to ultimately be able to raise the operating temperatures and increase the rate of both cellulose hydrolysis and sugar conversion compared to the rate in the smaller container. “We haven’t yet gotten the rates up to what they were in the [smaller] pressurized reactor, but we are closing in on that goal,” Lane says. It is very important to think about the details of the how the reaction works, including the type and amounts of materials used, and the mechanics of operating the reactor. “These are usually overlooked in the initial stages of research but become critical at larger scale,” Lane adds.

Big idea: The group is testing the new apparatus using sugars and cellulose, but once they’ve optimized the reactor they plan to use other biomass feedstocks such as Miscanthus, a perennial tall grass that has excellent potential as a feedstock.

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