Sustainable Bioenergy: Genomics and Biofuels Development

Believe it or not, genomics may be a key factor in addressing the global energy crisis. Through their knowledge in this field, researchers are developing a better understanding of how to harness various renewable energy sources, such as lignocellulosic biomass, microalgae, and cyanobacteria. Furthermore, it appears that genetic engineering of enzymes will be a key factor in optimizing development of sustainable biofuels that can someday replace fossil fuels on a global scale.

One of the primary dilemmas currently facing biofuel scientists involves the use of first-generation versus second-generation biofuels. First-generation biofuels, primarily consisting of ethanol and biodiesel, are derived from sugars, starches, and oils, and the crops used to create these fuels compete with food crops for the use of agricultural land and water. Moreover, although burning ethanol and biodiesel releases less carbon than burning petroleum, these biofuels are frequently created and processed in ways that harm the environment and can result in more deforestation, pollution, water use, and release of greenhouse gases than with fossil fuels (Fargione et al., 2008). As a result, scientists, politicians, and the public are looking to genomics to find new energy sources and ways to convert biomass into usable energy.

Efforts in this area have led to advances in second-generation biofuel production that focus on the extraction of energy from lignocellulosic biomass sources. Unlike the easily processed sugars and oils in first-generation biofuels, lignocellulosic biomass consists of matter composed of the woody, inedible parts of plants, such as grasses, crops, and forest waste. One advantage of using these materials is that they are not typically grown on agricultural land, thus removing competition between use of this land for food production and use of this land for fuel production.

So, how is energy stored and extracted from lignocellulosic biomass? First, by way of photosynthesis, plants convert solar energy and store this energy in their cell wall polymers in the forms of cellulose, hemicellulose, and lignin (Rubin, 2008). Cellulose, a chain of glucose molecules, is the main fuel component in lignocellulosic biomass, but unlike starch, it is not soluble in water. Another challenge in the use of lignocellulosic biomass is that its cellulose comes bonded to hemicellulose (which consists of five- and six-carbon polysaccharides) and lignin (which provides the plant with structure and natural defenses against insects and pathogens). Hemicellulose is more difficult to ferment than cellulose, but through genome-scale studies of the various enzymes present in yeasts and fungi, various cellulases and hemicellulases have been identified and used to successfully enhance the degradation of cellulose (Turner et al., 2007). Lignin, however, remains the primary obstacle to saccharification, as it is itself immune and protects both the cellulose and hemicellulose from enzymatic conversion. Lignocellulosic biomass is therefore more difficult to degrade than first-generation feedstocks like corn, sugarcane, soy, palm, or grapeseed, so researchers continue to seek methods for refining the process and improving yields of this plentiful and potentially inexpensive fuel source.

The main areas of genomic investigation in optimizing lignocellulosic biomass for biofuel production center on fuelstocks with modifiable traits, in which enzymatic activities can be engineered for biorefining (Sarath et al., 2007). C₄ perennial grasses, such as switchgrass, sorghum, and Miscanthus, present some of the best options for bioethanol feedstocks. For example, the U.S. Department of Energy's Bioenergy Feedstock Development Program identified switchgrass as one of the most promising energy feedstocks because of its yield rate, ability to grow from seed on marginal lands, capacity to protect soil from erosion, and potential to grow without fertilizer or replanting (Bouton, 2007). The Joint Genome Institute is currently sequencing the switchgrass genome, but researchers have already observed that although many genotypes of switchgrass exist, only two cultivars present options for efficient genetic engineering (Sticklen, 2008). Moreover, switchgrass cannot grow in colder climates, so trait analyses must be conducted for other biomass sources, such as C₃ plants like poplar and willow. Poplar was one of the first bioenergy sources to be sequenced, but researchers are still at work on maximizing poplar's speed of growth, thickness of stems, cell wall chemistry, branching tendencies, and reaction to competition for light (Rubin, 2008).

Many researchers also think that corn, or maize, could serve as a model for C₄ fuelstocks, and sequencing of the B73 corn cultivar is nearly complete. In February 2008, researchers at Washington University in St. Louis announced that they had released a draft blueprint, with the final genome to be completed by the end of 2008. Not only is maize the foremost ethanol fuelstock in the U.S., but it has long been a source for key scientific breakthroughs, such as the detection and cloning of transposable elements, the correlation of cytological and genetic crossing over, and the uncovering of several other genetic phenomena (Lawrence et al., 2008). Maize is a particularly attractive model for genetic modification of perennial grasses, because it is similar to switchgrass and Miscanthus in using the C₄ photosynthetic pathway, and it also originates in the same subfamily of the PACCAD clade (Panicoid, Arundinoid, Chloridoid, Centothecoid, Aristidoid, and Danthonioid lineages). Moreover, corn subspecies include diploids and tetraploids that are both annual and perennial, and finally, maize can be easily hybridized in a manner similar to Miscanthus (Lawrence & Walbot, 2007).

The chemical steps for creating biofuels from lignocellulose include the collection of the biomass, the breakdown of the polymers into sugars, and the conversion of the sugars into ethanol or another biofuel (Figure 1). Methods of extracting energy from lignocellulosic biomass generally involve the application of heat and/or acid treatments to separate the lignin from the cellulose and hemicellulose; one of the most common techniques includes enzymatic hydrolysis, fermentation, and distillation. Right now, the process is expensive and inefficient, and it produces fuel with a lower energy content than is scalable on an industrial level. Recently, however, scientists have successfully combined the hydrolysis and fermentation stages in a process called simultaneous saccharification and fermentation (Kristensen et al., 2008). Of course, there are still many challenges with this process, including finding favorable temperature and pH conditions and recycling the fermenting organism and enzymes, among others (Oloffson, 2008).

In order to improve the biorefining process for cellulosic ethanol, researchers are turning to the enzymes used by microbia in the guts of wood-feeding termites and other organisms that naturally process lignocellulosic biomass. Various microbes from within termite mid- and hindguts have so far been difficult to sequence, but efforts are underway to isolate and identify those enzymes that are responsible for hydrolysis and fermentation of cellulosic materials. For instance, in a paper published in the November 22, 2007, issue of Nature, Falk Warnecke and his team provide a metagenomic analysis of the bacteria in the hindgut of a Nasutitermes termite and distinguish the bacterial genes that can perform complex enzymatic hydrolysis (Warnecke et al., 2007). In addition to the hundreds of genes that help digest lignocellulose, researchers have also discovered that Nasutitermes' hindgut appears particularly dominated by two bacteria (fibrobacters), which deconstruct lignocellulose, and treponomes that ferment sugar (Figure 2; Potera, 2008). By applying these data and conducting other research into the digestive enzymes present in organisms that can break down and convert lignocellulose, scientists hope to reduce the lignin content in feedstocks without exposing plants to environmental threats such as diseases and insects, as well as to design more efficient enzymes that can streamline commercial-scale biorefining processes.

Of course, the termite is not the only source of model enzymes, nor is ethanol the only biofuel to which researchers are applying genomics. Many problems still exist with ethanol, even that derived from lignocellulosic biomass, including lower energy efficiency per unit than petroleum and high water solubility, which results in corrosion of pipelines and vehicle engines (Hill et al., 2006). Biobutanol, an alcohol that is also derived from lignocellulosic biomass, is another potential major fuel source. The conversion process for butanol involves creating a synthesis gas from the biomass and then using a catalyst for conversion to butanol. Researchers have identified, among others, Clostridium acetobutylicum as a promising catalyst bacterium. In fact, scientists have sequenced this organism's genome and are currently experimenting with recombinant strains to improve the bacterium's production levels (Dürre, 2007). In addition, E. coli has been genetically modified to create butanol, but it still produces lower energy yields than ethanol (Wackett, 2008). As with all of the biofuels under discussion, optimal yield and conversion conditions have not yet been reached, but many researchers believe that biobutanol may be more viable than bioethanol as a long-term fuel source.

Beyond second-generation biofuels, scientists are also working with so-called third-generation biofuels, which are derived from microalgae and cyanobacteria; however, this field has not yet been explored as extensively as lignocellulosic biomass. As photosynthetic organisms, microalgae convert sunlight, water, and carbon dioxide to their own oil, which is then converted to biodiesel through existing refining processes (Figure 3). Advantages to this process include the facts that microalgae is an aquaculture, that it reproduces rapidly, that it can grow in salt water, that it can generate biomass with a higher energy content than ethanol, and that the resulting biofuel can be used as a pure product in existing transport systems (Holzman, 2008). The actual amount of oil that is produced, however, depends upon such critical factors as sunlight and CO₂ levels, resulting in the need for further research related to land-use options for pond farms and development of costly and as-yet imperfect photobioreactors. However, genetic engineering of microalgae could result in an increased photosynthetic and biomass yield, advanced growth rates, increased oil content in the biomass, and regulated temperature tolerance, which is a current challenge (Chisti, 2007).

Biohydrogen is another fuel that can be developed from both microalgae and cyanobacteria (formerly known as blue-green algae), and it has the advantage of being a completely environmentally clean fuel. Today's biohydrogen is still created by processes that rely on fossil fuels and release greenhouse gases into the atmosphere. However, as early as 1939, a researcher named Hans Gaffron at the University of Chicago discovered that unicellular green microalgae could produce hydrogen, and since then, hydrogenases have been discovered across the prokaryotic and eukaryotic realms (Kruse et al., 2005). Similarly, microalgae and cyanobacteria have hydrogenases that use solar energy to split water (biophotolysis) and generate hydrogen without releasing any carbon dioxide. However, one major obstacle under examination is that the presence of oxygen tends to generate lower yields of hydrogen, so genetic engineering will be necessary to maximize hydrogen production while adjusting the hydrogenase's tolerance to oxygen.

These are just a few of the ways that genomics is influencing the development of sustainable bioenergy sources and conversion processes, and many other genetic solutions have yet to be explored. In the coming years, researchers will continue to look to nature for solutions to the global energy crisis. By applying genomic research and engineering to renewable fuelstocks and the bacteria and enzymes that convert those sources to energy, scientists can optimize billions of years of evolution to meet our growing energy needs in an environmentally friendly way.