The Sol-Gel Process - Ígor, Notas de estudo de Engenharia Elétrica
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The Sol-Gel Process - Ígor, Notas de estudo de Engenharia Elétrica

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REVIEWS

Genomics of cellulosic biofuels Edward M. Rubin1,2

The development of alternatives to fossil fuels as an energy source is an urgent global priority. Cellulosic biomass has the potential to contribute to meeting the demand for liquid fuel, but land-use requirements and process inefficiencies represent hurdles for large-scale deployment of biomass-to-biofuel technologies. Genomic information gathered from across the biosphere, including potential energy crops and microorganisms able to break down biomass, will be vital for improving the prospects of significant cellulosic biofuel production.

T he capture of solar energy through photosynthesis is a pro- cess that enables the storage of energy in the form of cell wall polymers (that is, cellulose, hemicellulose and lignin). The energy stored in these polymers can be accessed in a variety

of ways, ranging from simple burning to complex bioconversion processes. The high energy content and portability of biologically derived fuels, and their significant compatibility with existing pet- roleum-based transportation infrastructure, helps to explain their attractiveness as a fuel source. Despite the increasing use of biofuels such as biodiesel and sugar- or starch-based ethanol, evidence sug- gests that transportation fuels based on lignocellulosic biomass rep- resent the most scalable alternative fuel source1. Lignocellulosic biomass in the form of plant materials (for example, grasses, wood and crop residues) offers the possibility of a renewable, geograph- ically distributed and relatively greenhouse-gas-favourable source of sugars that can be converted to ethanol and other liquid fuels. Calculations of the productivity of lignocellulosic feedstocks, in part based on their ability to grow on marginal agricultural land, indicates that they can probably have a large impact on transportation needs without significantly compromising the land needed for food crop production2.

Lignocellulosic biofuel production involves collection of biomass, deconstruction of cell wall polymers into component sugars (pre- treatment and saccharification), and conversion of the sugars to biofuels (fermentation) (Fig. 1). Partially because of the historically low demand for biologically based transportation fuels, each step in this process is in the early stages of optimization for efficiency and throughput. The crops from which biomass is currently derived have not been domesticated for this particular purpose and the present methods for saccharification and fermentation are inefficient and

expensive. However, the recent and pressing desire to develop alter- natives to fossil fuels has made the rapid improvement of biofuel production a high priority, in which biologically derived energy (‘bioenergy’)-relevant genomic insights and resources will have an important role (Table 1).

Biomass From the perspective of transportation fuels, plants can be viewed as solar energy collectors and thermochemical energy storage systems. It is the storage of energy in a form that can later be accessed via thermochemical or enzymatic conversion that distinguishes biomass from other renewable energy sources. Cellulosic biomass, sometimes referred to as lignocellulosic biomass, is an abundant renewable resource that can be used for the production of alternative trans- portation fuels3. The three main components of lignocellulose are cellulose, hemicellulose and lignin (Fig. 2), with the relative propor- tions of the three dependent on the material source4. Cellulose, the main structural component of plant cell walls, is a long chain of glucose molecules, linked to one another primarily by glycosidic bonds5. Hemicellulose, the second most abundant constituent of lignocellulosic biomass, is not a chemically well defined compound but rather a family of polysaccharides, composed of different 5- and 6-carbon monosaccharide units, that links cellulose fibres into microfibrils and cross-links with lignin, creating a complex network of bonds that provide structural strength5. Finally lignin, a three- dimensional polymer of phenylpropanoid units, can be considered as the cellular glue providing the plant tissue and the individual fibres with compressive strength and the cell wall with stiffness6, in addition to providing resistance to insects and pathogens.

1DOE Joint Genome Institute, 2800 Mitchell Drive, Walnut Creek, California 94598, USA. 2Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, California 94720, USA.

Solar energy

Feedstock

Sugars

Biofuels

Fuel-producing microorganisms

Physical pre-treatment, chemicals and enzymes

Figure 1 | Biology of bioconversion of solar energy into biofuels. Solar energy is collected by plants via photosynthesis and stored as lignocellulose. Decomposition of the cellulosic material into simple 5- and 6-carbon sugars is achieved by physical and chemical pre- treatment, followed by exposure to enzymes from biomass-degrading organisms. The simple sugars can be subsequently converted into fuels by microorganisms.

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As we can retrospectively view the features that made certain wild plants desirable for domestication thousands of years ago to become today’s food crops, we are now prospectively defining criteria to choose plants with potential to serve as dedicated bioenergy crops in the future. These include cell wall composition, growth rate, suit- ability for growth in different geographical regions, and resource-use efficiencies. With these features in mind, a list of potential bioenergy crops is being developed and targeted for different growing condi- tions7. Most plants assimilate their CO2 first into a C3 compound, whereas a smaller subset use a C4 compound. Plants using C4 pho- tosynthesis tend to be among the most productive, having higher maximum efficiencies of light, nitrogen and water use in assimilating carbon. The C4 group of potential energy crops includes various perennial grasses such as switchgrass and Miscanthus. These grasses have the advantages of not requiring replanting after a yearly harvest, rapid growth, high biomass density per unit area, and low nutrient and water needs, enabling growth on marginal agricultural land. Disadvantages are that C4 plants are rare in cold climates and unable to grow at temperatures below 10 uC. In these environments, trees, which exclusively depend on C3 photosynthesis, provide the only

candidate species. The C3 group of potential energy crops includes trees, such as poplar and eucalyptus, which have relatively rapid growth potential in difficult-to-plough environments. It is highly likely that multiple different energy feedstocks will be deployed depending on latitude, geography, water availability and landowner acceptance.

Until recently, minimal effort has been directed towards optim- izing potential energy crops for the generation of transportation fuels. This is in stark contrast to the agronomic development of food crops, which have been domesticated for thousands of years to max- imize productivity. Teosinte, the wild precursor to modern maize, was first recognized by Native Americans more than 5,000 years ago as a potential food crop. The domestication of teosinte resulted in its conversion from a wild plant, the characteristics of which had been orchestrated by natural selection maximizing survival and reproduc- tion, into a plant whose morphology and physiology had been exten- sively altered by artificial selection to increase its nutritional yield and ease of harvest8. More recently, selective breeding as well as agro- nomic advances have resulted in improvement over several orders of magnitude in the nutritional value per acre of modern maize

Table 1 | Bioenergy genomes

Organism Genome size (megabases)

Status Reference

Feedstocks and feedstock models Populus trichocarpa (poplar) 480 Complete Ref. 9 Chlamydomonas reinhardtii 120 Complete Ref. 34 Glycine max (soya bean) 1,200 Draft – Manihot esculenta (cassava) 770 In progress – Sorghum bicolor 760 In progress – Eucalyptus globulus 600 In progress – Brachypodium distachyon 355 In progress – Zea mays (maize) 2,500 In progress – Elaeis guineensis (oil palm) ,3,400 In progress http://www.checkbiotech.org/green_News_Biofuels.aspx?infoId515100 Panicum virgatum (switchgrass) ,5,600 In progress – Setaria italica (foxtail millet) ,515 In progress – Biomass degraders Acidothermus cellulolyticus 11B 2.4 Complete – Bacillus pumilis SAFR-032 3.7 Complete Ref. 35 Caldicellulosiruptor saccharolyticus DSM 8903 3.0 Complete – Clostridium phytofermentans ISDg 4.8 Complete – Clostridium thermocellum ATCC 27405 3.8 Complete – Cytophaga hutchinsonii ATCC 33406 4.4 Complete – Flavobacterium johnsoniae UW101 6.1 Complete – Rubrobacter xylanophilus DSM9941 3.2 Complete – Saccharophagus degradans 5.1 Complete Ref. 36 Thermobifida fusca strain YX 3.6 Complete Ref. 37 Clostridium cellulolyticum H10 4.0 Draft – Elusimicrobium minutum Pei191 1.6 Draft – Nectria haematococca/Fusarium solani 51 Draft – Phanerochaete chrysosporium 35.1 Draft – Postia placenta 33 Draft – Sagittula stellata E-37 5.3 Draft – Trichoderma reesei/Hypocrea jecorina 33 Draft – Cellulomonas flavigena DSM 20109 ,4.0 In progress – Cellvibrio japonicus Ueda107 ,6.0 In progress – Fibrobacter succinogenes subsp. succinogenes S85 ,3.8 In progress – Ruminococcus albus 4.0 In progress – Teredinibacter turnerae T7902 ,2 In progress – Termite hindgut community NA Complete Ref. 23 Poplar biomass degrading community NA In progress http://www.jgi.doe.gov/sequencing/lspssseqplans2007.html Asian longhorned beetle (Anoplophora glabripennis) gut community

NA In progress http://www.jgi.doe.gov/sequencing/DOEmicrobes2007.html

Bovine rumen community transcriptome NA In progress http://www.energybiosciencesinstitute.org/ index.php?option5com_content&task5view&id5159&Itemid520

Fuel producers Clostridium acetobutylicum ATCC 824 4.0 Complete Ref. 38 Clostridium beijerinckii NCIMB 8052 6.0 Complete – Pichia stipitis 15.4 Complete Ref. 27 Thermoanaerobacter tengcongensis MB4 2.7 Complete Ref. 39 Zymomonas mobilis subsp. mobilis ZM4 2.1 Complete Ref. 40 Bacillus coagulans 36D1 2.9 Draft – Thermoanaerobacter pseudethanolicus 39E 2.4 Draft – Clostridium ljungdahlii ,4.0 In progress –

Bioenergy-relevant organisms for which large-scale genome projects have been completed or are under way are listed. Information on genome projects without references can be found at http:// www.ncbi.nlm.nih.gov/sites/entrez?db5genomeprj.

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compared to that of teosinte. Some of the most rapid increases have occurred in the past 40 years, both from advances in agronomic practices and, importantly, from the application of modern genetics. The optimization of bioenergy crops as feedstocks for transportation fuels is in its infancy, but already genomic information and resources are being developed that will be essential for accelerating their domestication. Many of the traits targeted for optimization in poten- tial cellulosic energy crops are those that would improve growth on poor agricultural lands, to minimize competition with food crops over land use.

Populus trichocarpa (poplar), the first tree and potential bioenergy crop to have its genome sequenced (Table 1)9, illustrates some of the issues and potential of applying genomics to the challenge of optim- izing energy crops. The traits for which the genetic underpinnings will be sought in the genomes of bioenergy-relevant plants, such as poplar, include those affecting growth rates, response to competition for light, branching habit, stem thickness and cell wall chemistry. Significant effort will go into maximizing biomass yield per unit land area, because this more than any other factor will minimize the impact on overall land use. One can imagine trees optimized to have short stature to increase light access and enable dense growth, large stem diameter, and reduced branch count to maximize energy den- sity for transport and processing. Trees have evolved with highly rigid and stable cell walls due to heavy selective pressure for long life and an upright habit. Plants domesticated for energy production, with a

crop cycle time of only a few years, would have less need for a rigid cell wall than wild plants with lifetimes of a hundred years or more. Alterations in the ratios and structures of the various macromole- cules forming the cell wall are a major target in energy crop domest- ication to facilitate post-harvest deconstruction at the cost of a less rigid plant.

Already, by comparing several of the presently available plant gen- omes (poplar9, rice10,11, Arabidopsis12; see Table 1) coupled with large- scale plant gene function and expression studies, a number of can- didate genes for domestication traits have been identified13,14. These include many genes involved in cellulose and hemicellulose synthesis as well as those believed to influence various morphological growth characteristics such as height, branch number and stem thickness15. In addition to homology-based strategies, other genome-enabled strategies for identifying domestication candidate genes are being used. These include quantitative trait analysis of natural variation and genome-wide mutagenesis coupled with phenotypic screens for traits such as recalcitrance to sugar release, acid digestibility and general cell wall composition. The availability of high-through- put transgenesis in several plant systems16 will facilitate functional studies to determine the in vivo activities of the large number of domestication candidate genes. Using these strategies, genes affecting features such as plant height, stem elongation and trunk radial growth, drought tolerance, and cell wall stability are but a few of the features that are likely to be identified as targets for domestication

OH

Macrofibril

Plant cell

Plant

Cell wall

Lignin

Lignin

Hemicellulose

Pentose

Crystalline cellulose

Hydrogen bondCellodextrin

n-3

n-3

n-3

n-3

n-3

Glucose

Hexose

10–20 nm

Macrofibril

OH

OH

OH

OH

p-Coumaryl alcohol Coniferyl alcohol Sinapyl alcohol

OH

O

H G S

OO

Figure 2 | Structure of lignocellulose. The main component of lignocellulose is cellulose, a b(1–4)-linked chain of glucose molecules. Hydrogen bonds between different layers of the polysaccharides contribute to the resistance of crystalline cellulose to degradation. Hemicellulose, the second most abundant component of lignocellulose, is composed of various 5- and 6-carbon sugars such as arabinose, galactose, glucose, mannose and xylose. Lignin is composed of three major phenolic components, namely

p-coumaryl alcohol (H), coniferyl alcohol (G) and sinapyl alcohol (S). Lignin is synthesized by polymerization of these components and their ratio within the polymer varies between different plants, wood tissues and cell wall layers. Cellulose, hemicellulose and lignin form structures called microfibrils, which are organized into macrofibrils that mediate structural stability in the plant cell wall.

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in a fraction of the time required to carry out similar studies unaided by the plant genomes and genomic approaches17.

Biomass degradation The breakdown of biomass involves the release of long-chain poly- saccharides, specifically cellulose and hemicellulose, and the sub- sequent hydrolysis of these polysaccharides into their component 5- and 6-carbon chain sugars. Early chemical processes for cellulose degradation depended heavily on acid treatments5,18, and even today industrial processes for biomass degradation involve heat and acidic conditions and tend to be expensive, slow and relatively inefficient19. Furthermore, some of these pre-treatments produce inhibitors (that is, furfural and 5-hydroxymethylfurfural) that decrease the overall yield of the fermentation process.

The human need for efficient breakdown of lignocellulosic bio- mass for biofuel production is relatively new; however, a variety of organisms have evolved to take advantage of this nutrient source, including the free-living organisms and symbiotic animal–microbe consortia invariably present in biomass-rich environments. Increasing our knowledge of the biochemical machinery used by these organisms for the breakdown of biomass offers new avenues for the development of biologically based processes that could poten- tially accomplish biomass conversion at an industrial scale. Just as plant genomes are providing us with new approaches for accelerating feedstock domestication, genomics of biomass-degrading microbes and microbial consortia offer a new means to enzyme discovery.

Microbial strategies for degrading lignocellulose are diverse, yet our current understanding of the enzymes involved in these processes is limited to a handful of model organisms such as the fungus Trichoderma reesei and the bacterium Clostridium thermocellum20. Recent genome sequencing of these two biomass degraders, as well as a host of other cellulolytic species (Table 1), has expanded our repertoire of known or potential cellulolytic enzymes. The knowledge of biomass degradation pathways is soon to be increased even more by a number of large-scale genomic surveys under way of isolated organisms and microbial communities known to degrade biomass (Table 1).

Considering the current dependence on acid and heat pre-treat- ment in the deconstruction of lignocellulose, it is clear that enzymes that are stable and active at low pH values and at high temperatures are of particular value. Thus, enzymes derived from thermophilic and acidophilic organisms known to degrade lignocellulose, such as Caldicellulosiruptor saccharolyticus and Acidothermus cellulolyticus, hold significant promise for industrial processes21. However, the number of sequenced thermoacidophilic organisms is low, partly because of the significant obstacles in cultivating these organisms in the laboratory.

Many novel enzymes and enzyme systems that have evolved to make use of cellulosic biomass are present in difficult-to-culture microbes22. This is particularly the case for the communities of microbes inhabiting the guts of lignocellulose-consuming insects and ruminants. Recently, in response to the challenge of studying difficult-to-culture organisms, metagenomic approaches have been developed to access the information encoded in their genomes (Table 1). This typically involves the high-throughput sequencing of DNA extracted directly from the mixtures of organisms present in an environment.

The metagenomic analysis of a wood-feeding higher termite23 has recently revealed hundreds of genes encoding carbohydrate-active enzymes, many of which were subsequently shown to be biochemically active. From the perspective of an industrial application, the termite hindgut is too complex a model to mimic because it comprises hun- dreds of microbial species and lignocellulose-degrading enzymes. However, termites tend to feed on complex diets of several plant species, which translate to more complex hindgut communities and enzyme inventories. Thus, examination of this single community reveals enzymes capable of hydrolysing a broad assortment of chemical

bonds. The saccharification of a single uniform cellulosic feedstock, particularly one engineered to have a simplified cell wall, will presum- ably require fewer enzymes and less downstream optimization of gene regulation and enzyme activity in a production host.

The availability of a wide range of naturally occurring lignocellu- lose-degrading enzymes increases the chances of successful enzyme optimization for industrial processes. Optimization of the sacchar- ification process is crucial because the cost of cellulases remains a key barrier to economical production of biofuels3. A more diverse set of candidate enzymes—identified through a combination of conven- tional cultured microbial genome studies coupled with envir- onmental prospecting methods such as metagenomics—improves the likelihood of obtaining enzymes with activities and stability sui- ted to a variety of industrial processes.

Biofuel production

Although the final steps of cellulosic ethanol production will require much of the same infrastructure developed for the production of sugar- and starch-based ethanol, changes will need to be made to exploit the diversity of sugars generated from the breakdown of bio- mass. Whereas the conversion of starch-based feedstock results prim- arily in hexoses, the products from degradation of lignocellulosic biomass, composed in part of hemicellulose, include large amounts of the pentose sugars D-xylose and L-arabinose. In contrast to the hexose sugars, the pentose sugars cannot be fermented by wild-type Saccharomyces cerevisiae24. Another factor limiting ethanol yield is the toxicity of ethanol to the fermenting host. Most fermenting organisms such as S. cerevisiae cannot tolerate ethanol concentrations exceeding 25% (v/v)25, resulting in a product that must then be con- centrated through distillation. Distillation represents an expensive and energy-intensive step in ethanol production.

These limitations in ethanol production have generated significant interest in developing new organisms able to exploit fully the break- down products of lignocellulosic biomass as well as tolerate the pro- ducts of fermentation26. Pichia stipitis represents one yeast species of relevance to biofuel research based on its natural ability to ferment the pentose sugar xylose. Its recently sequenced genome revealed insights into the metabolic pathways responsible for this process27, and investigators are already working on the optimization of this pathway in P. stipitis as well as on the construction of systems for the heterologous expression of P. stipitis genes. Escherichia coli has already been genetically engineered for the conversion of all hexose and pentose sugars present in hemicellulose polymers28. The result- ing genetically engineered strain has an ethanol production rate sim- ilar to yeast29, and its ethanol tolerance has been increased by selection on enrichment media30. In the future, genomics and path- way engineering should considerably facilitate the development of a variety of organisms able to use the full repertoire of cellulosic and hemicellulosic sugars and tolerate high ethanol concentrations to optimize ethanol yields.

Although ethanol production facilities and distribution centres are rapidly increasing in number, ethanol as a transportation fuel has several disadvantages that encourage the pursuit of better suited alternative biofuels in the future. Disadvantages of ethanol as a bio- fuel include: (1) high solubility of ethanol in water, which necessitates an energy-intensive distillation step; (2) diminished energy per gal- lon content of ethanol compared to petroleum; (3) difficulties in the distribution of ethanol via today’s pipeline infrastructure owing to its hygroscopic nature; and (4) incompatibilities with many current vehicles at higher blending volumes. Thus, work is under way to enable the synthesis of next-generation biofuel products, such as higher-chain alcohols, alkanes, and other molecules with structures and activities closer to that of petroleum and diesel. Butanol, for example, can be used in conventional engines either in pure form or mixed with petroleum, can be transported by existing pipelines, and has a higher energy content than petroleum31. A number of organisms, such as Clostridium acetobutylicum, produce butanol

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from sugars via the ABE (acetone, butanol, ethanol) fermentation and can be manipulated to produce mostly butanol31. E. coli has recently been engineered to produce isobutanol and other alcohols via a non-fermentative pathway that may be more readily adapted to large-scale production, using heterologous expression of Lactococcus lactis and Bacillus subtilis genes32. The genomics-enabled construc- tion of partial or fully artificial biological systems through ‘synthetic biology’ approaches will be key in developing efficient, inexpensive biofuel production systems33.

Summary This perspective has focused on biofuels derived from lignocellulosic feedstocks; however, another potential source of transportation fuels is biodiesel, derived from oil-producing plants and microbes. Predominant sources of biodiesel presently include soya bean, rape- seed and palm oils. The genomes of soya bean and oil palm are both currently being sequenced (Table 1), information from which will be called on to increase oil production and facilitate plant growth in a broader range of habitats. Oil-producing algae are also under active study as an alternative source for biodiesel production. One of the major advantages of algae is their growth in liquid, negating the issue of potential competition between food and energy crops for land use. Chlamydomonas reinhardtii is one of a series of algal species whose genomes are becoming available for analysis34. Considering the mag- nitude of the transportation fuel problem, biodiesel is likely to con- tribute at least in part to the solution, and genomic research on oil producers will aid in making biodiesel production cheaper and more efficient.

Placing a man on the Moon and sequencing the human genome represented large-scale technologically challenging projects. In both these instances, substantial research and development efforts resulted in technological advances unforeseen at the projects’ initiation. The strategies that were pioneered in sequencing and interpreting the human genome for the improvement of human health are now poised to be an important contributing technology in the challenge to develop environmentally and socially acceptable alternatives to fossil fuels.

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Acknowledgements I would like to thank S. Tringe, M. Hess, J. Tuskan, P. Hugenholtz, J. Bristow, B. Simmons, S. Long, J. Fruchart-Najib and H. Blanch for their input to the manuscript. This work was performed under the auspices of the US Department of Energy’s Office of Science, Biological and Environmental Research Program, and by the University of California, Lawrence Berkeley National Laboratory under contract number DE-AC02-05CH11231, Lawrence Livermore National Laboratory under contract number DE-AC52-07NA27344, and Los Alamos National Laboratory under contract number DE-AC02-06NA25396.

Author Information Reprints and permissions information is available at www.nature.com/reprints. Correspondence should be addressed to E.M.R. (emrubin@lbl.gov).

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