Review Cel Plants to power:bioenergy to fuel the future Joshua S.Yuan1.2.3,Kelly H.Tiller4,Hani Al-Ahmad1.5,Nathan R.Stewart1.2 and C.Neal Stewart Jr1 1 Department of Plant Sciences,University of Tennessee,Knoxville,TN 37996,USA 2 University of Tennessee Institute of Agriculture Genomics Hub,University of Tennessee,Knoxville,TN 37996,USA 3 Current address:Institute for Plant Genomics and Biotechnology,Texas A&M University,College Station TX 77843,USA 4 Department of Agricultural Economics and the Office of Bioenergy Programs,University of Tennessee,Knoxville,TN 37996,USA 5 Department of Biology Biotechnology,An-Najah National University,Nablus,West Bank,Palestine Bioenergy should play an essential part in reaching tar- is also an important raw product in the chemical industry. gets to replace petroleum-based transportation fuels with Therefore,ethanol production has a particularly important a viable alternative,and in reducing long-term carbon role in transforming petroleum-based economies to bio- dioxide emissions,if environmental and economic sus- mass-based sustainable and environment-friendly econ- tainability are considered carefully.Here,we review omies. different platforms,crops,and biotechnology-based improvements for sustainable bioenergy.Among the Ethanol processing platforms different platforms,there are two obvious advantages Ethanol can be produced using agricultural products such as to using lignocellulosic biomass for ethanol production: starch and sugar,or lignocellulosic biomass(see Glossary; higher net energy gain and lower production costs.How- Figure la and b).Currently >10 billion gallons ofethanol is ever,the use of lignocellulosic ethanol as a viable alterna- produced globally per year from starch(maize)and sugar tive to petroleum-based transportation fuels largely (sugarcane and sugar beet)through mature industrialized depends on plant biotechnology breakthroughs.We procedures,including hydrolysis of starch and fermentation examine how biotechnology,such as lignin modification, ofsugar(Figure la)[3,6].Starch and sugar-based ethanol is abiotic stress resistance,nutrition usage,in planta expres- often referred to as a first-generation biofuel. sion of cell wall digestion enzymes,biomass production, feedstock establishment,biocontainment of transgenes, metabolic engineering,and basic research,can be used to Glossary address the challenges faced by bioenergy crop pro- Agriculture residuals (or residues):straw or 'stover'that are left in the field duction after harvest,or forest product 'waste'such as woodchips. Bioenergy feedstock:either the biomass crops themselves or the raw material Multiple choices for bioenergy that is input into the biorefinery. Biofuel cells:various electrochemical systems that can generate a current with Bioenergy refers to renewable energy from biological the electron or proton donated from microorganisms,often through oxidation sources that can be used for heat,electricity and fuel, reaction. and their co-products.There has been a resurgence of Carbon balance:also known as carbon dioxide balance;calculated as carbon dioxide emitted by biomass production and usage subtracted from the carbon interest in bioenergy recently,and several articles have dioxide fixed in the plant material,both above ground and underground.Thus, already addressed the potential impact of biotechnology on a negative carbon balance is desirable. renewable energy [1-5].However,in this review we will Fermentation:the conversion of sugars into ethanol by microorganisms under anaerobic conditions. integrate several of the key components of bioenergy, Greenhouse effect (also known as global warming):worldwide rise in including feedstock,processing platforms,enabling bio- temperature caused by particula ch as methane and carbon dioxide, technologies,ecological effects and economics,to gauge trapping heat from the sun on the Earth's surface.These gases are therefore called 'greenhouse gases' how plant biotechnology might impact bioenergy efficiency Lignocellulosic biomass:plants grown for ethanol production using the entire and sustainability.We will discuss the crucial choices of aboveground biomass.'Lignocellulosic'refers to the plant biomass that is composed of cellulose,hemicellulose and lignin polymers.Biomass can be feedstock (e.g.starch,sugar,fatty acid or cellulose)and hydrolyzed and resulting sugars can be for ethanol production and energy product (e.g.ethanol,biodiesel and others),the potentially other biofuels. economic feasibility and the pros and cons of different Net energy balance (NEB):the difference between the energy output and the energy input for biomass production and processing. choices,and the major technical breakthroughs needed Net energy ratio(NER):an altemative measure of energy gain consisting of the to develop a sustainable bioenergy industry. ratio of the energy output the energy nput for biomass production and processing. Pretreatment:an initial physical or chemical treatment to disassemble cell wall Choices of platforms components,typically involving factors such as high temperature and extreme In terms of modern bioenergy,ethanol,biodiesel and bio- pH. Recalcitrance:resista nce gas are the three major bioenergy products.Ethanol and of pla ell drolysis for the release of fermentable sugars. biodiesel can be used as transportation fuels,and ethanol Saccharification:the release of products such as cellulobiose and glucose from cellulose via chemical hydrolysis or enzymatic reactions. Corresponding author:Stewart Jr,C.N.(nealstewart@utk.edu). 1360-1385/$-see front matter 2008 Elsevier Ltd.All rights reserved.doi:10.1016/j.tplants.2008.06.001 Available online 16 July 2008 421
Plants to power: bioenergy to fuel the future Joshua S. Yuan1,2,3 , Kelly H. Tiller4 , Hani Al-Ahmad1,5 , Nathan R. Stewart1,2 and C. Neal Stewart Jr1 1 Department of Plant Sciences, University of Tennessee, Knoxville, TN 37996, USA 2 University of Tennessee Institute of Agriculture Genomics Hub, University of Tennessee, Knoxville, TN 37996, USA 3Current address: Institute for Plant Genomics and Biotechnology, Texas A&M University, College Station TX 77843, USA 4 Department of Agricultural Economics and the Office of Bioenergy Programs, University of Tennessee, Knoxville, TN 37996, USA 5 Department of Biology & Biotechnology, An-Najah National University, Nablus, West Bank, Palestine Bioenergy should play an essential part in reaching targets to replace petroleum-based transportation fuelswith a viable alternative, and in reducing long-term carbon dioxide emissions, if environmental and economic sustainability are considered carefully. Here, we review different platforms, crops, and biotechnology-based improvements for sustainable bioenergy. Among the different platforms, there are two obvious advantages to using lignocellulosic biomass for ethanol production: higher net energy gain and lower production costs. However, the use of lignocellulosic ethanol as a viable alternative to petroleum-based transportation fuels largely depends on plant biotechnology breakthroughs. We examine how biotechnology, such as lignin modification, abiotic stress resistance, nutrition usage,in plantaexpression of cell wall digestion enzymes, biomass production, feedstock establishment, biocontainment of transgenes, metabolic engineering, and basic research, can be used to address the challenges faced by bioenergy crop production. Multiple choices for bioenergy Bioenergy refers to renewable energy from biological sources that can be used for heat, electricity and fuel, and their co-products. There has been a resurgence of interest in bioenergy recently, and several articles have already addressed the potential impact of biotechnology on renewable energy [1–5]. However, in this review we will integrate several of the key components of bioenergy, including feedstock, processing platforms, enabling biotechnologies, ecological effects and economics, to gauge how plant biotechnology might impact bioenergy efficiency and sustainability. We will discuss the crucial choices of feedstock (e.g. starch, sugar, fatty acid or cellulose) and energy product (e.g. ethanol, biodiesel and others), the economic feasibility and the pros and cons of different choices, and the major technical breakthroughs needed to develop a sustainable bioenergy industry. Choices of platforms In terms of modern bioenergy, ethanol, biodiesel and biogas are the three major bioenergy products. Ethanol and biodiesel can be used as transportation fuels, and ethanol is also an important raw product in the chemical industry. Therefore, ethanol production has a particularly important role in transforming petroleum-based economies to biomass-based sustainable and environment-friendly economies. Ethanol processing platforms Ethanol can be produced using agricultural products such as starch and sugar, or lignocellulosic biomass (see Glossary; Figure 1a and b). Currently >10 billion gallons of ethanol is produced globally per year from starch (maize) and sugar (sugarcane and sugar beet) through mature industrialized procedures, including hydrolysis of starch and fermentation of sugar (Figure 1a) [3,6]. Starch and sugar-based ethanol is often referred to as a first-generation biofuel. Review Glossary Agriculture residuals (or residues): straw or ‘stover’ that are left in the field after harvest, or forest product ‘waste’ such as woodchips. Bioenergy feedstock: either the biomass crops themselves or the raw material that is input into the biorefinery. Biofuel cells: various electrochemical systems that can generate a current with the electron or proton donated from microorganisms, often through oxidation reaction. Carbon balance: also known as carbon dioxide balance; calculated as carbon dioxide emitted by biomass production and usage subtracted from the carbon dioxide fixed in the plant material, both above ground and underground. Thus, a negative carbon balance is desirable. Fermentation: the conversion of sugars into ethanol by microorganisms under anaerobic conditions. Greenhouse effect (also known as global warming): worldwide rise in temperature caused by particular gases, such as methane and carbon dioxide, trapping heat from the sun on the Earth’s surface. These gases are therefore called ‘greenhouse gases’. Lignocellulosic biomass: plants grown for ethanol production using the entire aboveground biomass. ‘Lignocellulosic’ refers to the plant biomass that is composed of cellulose, hemicellulose and lignin polymers. Biomass can be hydrolyzed and resulting sugars can be used for ethanol production and potentially other biofuels. Net energy balance (NEB): the difference between the energy output and the energy input for biomass production and processing. Net energy ratio (NER): an alternative measure of energy gain consisting of the ratio of the energy output and the energy input for biomass production and processing. Pretreatment: an initial physical or chemical treatment to disassemble cell wall components, typically involving factors such as high temperature and extreme pH. Recalcitrance: resistance of plant cell walls to hydrolysis for the release of fermentable sugars. Saccharification: the release of products such as cellulobiose and glucose from cellulose via chemical hydrolysis or enzymatic reactions. Corresponding author: Stewart Jr, C.N. (nealstewart@utk.edu). 1360-1385/$ – see front matter 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.2008.06.001 Available online 16 July 2008 421
Review Trends in Plant Science Vol.13 No.8 Starch Lignocellulosic biomass Methanol Plant lipid Hydrolysis Pretreatment Transesterification Glucose or fructose Disrupted biomass Biodiesel Sucrose Fermentation Hydrolysis Liquidification and pyrolysis Bioethanol Glucose,xylose and others Biomass (a) (c) Fermentation Biomass and other organic waste Bioethanol Anaerobic gasification (b) Biogases (d) TRENDS in Plant Science Figure 1.Processing platforms for producing different biofuels with various feedstocks.(a)Starch and sugar-based first generation ethanol.In this platform,starch can be hydrolyzed into monosaccharides,where sugar can be further fermented into ethanol.(b)Lignocellulosic ethanol.Lignocellulosic ethanol production involves pretreatment of biomass material,hydrolysis for monosaccharide production,and fermentation to produce ethanol.Lignocellulosic ethanol is believed to be the major component of the second generation of bioenergy.(c)Biodiesel.Biodiesel production often involves the transesterification of fatty acids.(d)Biomass gasification.Biomass can be used to produce methanol,carbon monoxide,hydrogen or other gases through a process of gasification. Even though the production of ethanol from starch nical hurdles can be scaled.Lignocellulosic feedstock can represents the most convenient and technically advanced be acquired from either dedicated biomass crops or forestry option for bioenergy in the USA,it would result in severe and agricultural residuals [5.9-131. competition between energy and food supplies,which is The key obstacle for transitioning from starch-based to probably not sustainable in the long term given that the lignocellulosic biofuels is the complicated structure of the net energy and carbon dioxide balance of the platform is cell wall,which is,by nature,resistant to breakdown-the not favorable (Table 1)[7,8].Therefore,in temperate recalcitrance problem.Current processes for lignocellulosic regions,biofuel (ethanol for now)production from ligno- biomass include pretreatment,saccharification (hydroly- cellulosic biomass represents the best choice if key tech- sis),and fermentation (Figure 1b)[14].Improvement or Table 1.Comparison of different platforms and bioenergy crops Platforms" Feedstock" NEB NERb C02 Annual Estab- Germ- Agricul. Ecological Refs GJ/ha/yr balance feedstock lishment plasm practiced benefits Ethanol from Maize 10-80 1.5-3.0 Positive Yes +++0 +++ +++ [4,28,30,31] starch or Sugarcane 55-80 3.0-4.0 Positive No +++ +++ +++ [4,28,30,311 sucrose Sugar beet 40-100 2.5-3.5 Positive Yes +++ +++ [28] Sweet 85-300 5-10 Positive Yes +++ ++ ++ [28,92] sorghum Ethanol from Miscanthus 250-550 15-70 Possibly Yes/No +++ [28 lignocellulosic negative feedstocks Switchgrass 150-500 10-50 Possibly No +++ [4,28,30,31.93 negative Poplar 150-250 10-20 Possibly No ++ ++ +++ [24,301 negative Biodiesel Soybean -20-10 0.2-0.6 Positive Yes ++ +++ +++ [28 Canola -5-2 0.7-1.0 Positive Yes +++ +++ +++ [28 Sunflower -10-0 0.3-0.9 Positive Yes +++ ++ +++ [28 Multiple platforms and crops are compared in a synthesis integrating information from multiple studies PAbbreviations:NEB,energy balance;NER,net energy ratio,which is the ratio of output to input energy needed to produce a fuel from a feedstock Favorable features are indicated by symbols,with +++being the most favorable. dAgricul.Practice,agricultural practice:how advanced is the current status of farming,harvesting,and processing. 422
Even though the production of ethanol from starch represents the most convenient and technically advanced option for bioenergy in the USA, it would result in severe competition between energy and food supplies, which is probably not sustainable in the long term given that the net energy and carbon dioxide balance of the platform is not favorable (Table 1) [7,8]. Therefore, in temperate regions, biofuel (ethanol for now) production from lignocellulosic biomass represents the best choice if key technical hurdles can be scaled. Lignocellulosic feedstock can be acquired from either dedicated biomass crops or forestry and agricultural residuals [5,9–13]. The key obstacle for transitioning from starch-based to lignocellulosic biofuels is the complicated structure of the cell wall, which is, by nature, resistant to breakdown – the recalcitrance problem. Current processes for lignocellulosic biomass include pretreatment, saccharification (hydrolysis), and fermentation (Figure 1b) [14]. Improvement or Figure 1. Processing platforms for producing different biofuels with various feedstocks. (a) Starch and sugar-based first generation ethanol. In this platform, starch can be hydrolyzed into monosaccharides, where sugar can be further fermented into ethanol. (b) Lignocellulosic ethanol. Lignocellulosic ethanol production involves pretreatment of biomass material, hydrolysis for monosaccharide production, and fermentation to produce ethanol. Lignocellulosic ethanol is believed to be the major component of the second generation of bioenergy. (c) Biodiesel. Biodiesel production often involves the transesterification of fatty acids. (d) Biomass gasification. Biomass can be used to produce methanol, carbon monoxide, hydrogen or other gases through a process of gasification. Table 1. Comparison of different platforms and bioenergy crops Platformsa Feedstocka NEBb GJ/ha/yr NERb CO2 balance Annual feedstock Establishment Germplasm Agricul. practiced Ecological benefits Refs Ethanol from starch or sucrose Maize 10–80 1.5–3.0 Positive Yes +++c +++ +++ + [4,28,30,31] Sugarcane 55–80 3.0–4.0 Positive No +++ +++ +++ + [4,28,30,31] Sugar beet 40–100 2.5–3.5 Positive Yes +++ ++ +++ + [28] Sweet sorghum 85–300 5–10 Positive Yes +++ ++ ++ ++ [28,92] Ethanol from lignocellulosic feedstocks Miscanthus 250–550 15–70 Possibly negative Yes/No + + + +++ [28] Switchgrass 150–500 10–50 Possibly negative No + + + +++ [4,28,30,31,93] Poplar 150–250 10–20 Possibly negative No + ++ ++ +++ [24,30] Biodiesel Soybean 20–10 0.2–0.6 Positive Yes ++ +++ +++ + [28] Canola 5–2 0.7–1.0 Positive Yes +++ +++ +++ + [28] Sunflower 10–0 0.3–0.9 Positive Yes +++ ++ +++ + [28] a Multiple platforms and crops are compared in a synthesis integrating information from multiple studies. b Abbreviations: NEB, energy balance; NER, net energy ratio, which is the ratio of output to input energy needed to produce a fuel from a feedstock. c Favorable features are indicated by + symbols, with +++ being the most favorable. d Agricul. Practice, agricultural practice: how advanced is the current status of farming, harvesting, and processing. Review Trends in Plant Science Vol.13 No.8 422
Review Trends in Plant Science Vol.13 No.8 replacement of these processes is crucial for increasing byproducts.Maize can be used as a bioenergy crop in two efficiency and for decreasing biofuel production costs ways:the starch in seeds can be used to produce ethanol, Obviating pretreatment along with simultaneous sacchar- and the crop residuals(termed stover)could potentially be ification and fermentation are two important factors that used to produce lignocellulosic ethanol.Sorghum (Sor- would decrease the cost oflignocellulosic ethanol production ghum bicolor)is the fifth most cultivated cereal crop in [14].Technology breakthroughs are badly needed. the world and is grown for grain,forage,sugar and fiber. Sorghum could also be used for bioenergy in several ways Biodiesel production processes Both the starch in the grain and the sugar could be feed- Biodiesel is a biofuel requiring far simpler processing tech- stock for ethanol fermentation using current technology nology compared with that required for ethanol.Biodiesel is platforms,and crop residuals could be useful for lignocel- a mixture of diesel fuel with oils from plant seeds,algae or lulosic ethanol production.Two features make sorghum a other biological sources such as animal renderings that have particularly attractive bioenergy crop.First,there would been transesterified for removal ofglycerol [15].A variety of not be strong competition between the use of land for food plant species are currently used for biodiesel production or for energy because the seeds can be used for food and including soybean,rapeseed and canola,sunflower and feed and the stems could be optimized for different plat- palm [15].After oil is extracted from plant organs it is forms of ethanol production,which is particularly import- transesterified,leading to biodiesel methyl or ethyl esters ant for heavily populated developing countries such as as products(Figure lc).Other potential choices for biodiesel China.Second,sorghum is drought and heat tolerant, include using terpenoid products from Copaifera species which would enable the usage of marginal land that is ('diesel tree')as biodiesel directly,or engineering the plant not suitable for the cultivation of many other crops [19]. terpenoid pathway to produce large amounts of sesquiter- Besides sorghum and maize,the residuals of other crops penes and diterpenes.As an alternative to diesel fuel, such as wheat and rice are also expected to be useful for biodiesel already has a niche in the current transportation lignocellulosic ethanol production. fuel system and is widely used,but production is relatively low.The sources and forms of biodiesel are diverse,and it is Sugar-producing plants important to consider the different environmental and Sugar can be used for direct fermentation of ethanol. economic factors that apply in the production of different Sugarcane(Saccharum officinarum)and sugar beet (Beta types of biodiesel [15].For example,feedstocks are as vulgaris)are the major sugar-producing plants.Sugar- diverse as soybean and cooking grease waste.Processing cane is adapted to warm temperate to tropical areas, feedstock to fuel is relatively simple,leading to widespread whereas sugar beet is grown in temperate areas.There- production but of variable quality. fore the two sugar crops occupy different geographical niches.Brazil is a successful example of a country that Biogas production process has reduced its gasoline usage by producing bioenergy. A third modern choice for bioenergy is biogas,which is The Brazilian national ethanol program,which is based produced from a variety of organic wastes,including plant solely on sugarcane,produces 4.2 billion gallons ofethanol straw,through gasification(Figure 1d).Biogases include a year [8),although the resultant ecological and environ- methane,hydrogen and carbon monoxide.Gasification mental effects are still debatable [20,211.Most ethanol using current technologies has a low net energy balance production using sugar beet takes place in Europe;how- and its usefulness therefore might be limited [16,17]. ever,using sugar beet to produce ethanol could potentially Besides the traditional biogases,hydrogen production by increase soil erosion and lower the net energy balance. green algae and microbes has been proposed as a potential Other sugar-producing crops include energy cane, source for a third generation biofuel [51.Unlike hydrogen improved cultivars of sugarcane and varieties of sweet production from other biomass sources,algae-based hydro- sorghum.All the above crops are annuals,with the excep- gen production uses a biological water-splitting reaction,in tion of sugarcane.Perennials are more desirable than which hydrogenase uses the photosynthetic electron trans- annuals as bioenergy feedstocks because they do not need port chain to reduce protons for hydrogen production [18] to be reseeded each growing season and therefore cultiva- Hydrogenase engineering for increased oxygen tolerance tion costs are lower. and systems biology research of genes and pathways involved in hydrogen production are needed to realize Dedicated bioenergy feedstocks the potential of this platform[18] Perennial bioenergy feedstocks are important sources of lignocellulosic biomass production.Switchgrass (Panicum Choices of crops and feedstocks virgatum)has been proposed as the major perennial feed- Bioenergy crops can be classified into the following four stock in the USA because it is widely adapted,has high groups:traditional cereal crops,traditional sugar-produ- biomass production,high C-4 photosynthetic efficiency, cing crops,dedicated lignocellulosic biomass feedstocks, and efficient use of water and nitrogen.Switchgrass and oilseed crops for biodiesel. yield is around 10 to 25 Mg/ha/yr depending on latitude, nutrition and other factors.Hybrid Miscanthus,including Traditional cereal crops Miscanthus x giganteus,is another highly favored Cereal crops are a major source for starch-based ethanol biomass feedstock,mainly in Europe.However,Mis- production.Maize(Zea mays)is an important food and feed canthus x giganteus is a sterile triploid clone that requires crop,used as processed food,oil,fodder,a vegetable and vegetative propagation.Similar to switchgrass,Mis- 423
replacement of these processes is crucial for increasing efficiency and for decreasing biofuel production costs. Obviating pretreatment along with simultaneous saccharification and fermentation are two important factors that would decrease the cost of lignocellulosic ethanol production [14]. Technology breakthroughs are badly needed. Biodiesel production processes Biodiesel is a biofuel requiring far simpler processing technology compared with that required for ethanol. Biodiesel is a mixture of diesel fuel with oils from plant seeds, algae or other biological sources such as animal renderings that have been transesterified for removal of glycerol [15]. A variety of plant species are currently used for biodiesel production including soybean, rapeseed and canola, sunflower and palm [15]. After oil is extracted from plant organs it is transesterified, leading to biodiesel methyl or ethyl esters as products (Figure 1c). Other potential choices for biodiesel include using terpenoid products from Copaifera species (‘diesel tree’) as biodiesel directly, or engineering the plant terpenoid pathway to produce large amounts of sesquiterpenes and diterpenes. As an alternative to diesel fuel, biodiesel already has a niche in the current transportation fuel system and is widely used, but production is relatively low. The sources and forms of biodiesel are diverse, and it is important to consider the different environmental and economic factors that apply in the production of different types of biodiesel [15]. For example, feedstocks are as diverse as soybean and cooking grease waste. Processing feedstock to fuel is relatively simple, leading to widespread production but of variable quality. Biogas production process A third modern choice for bioenergy is biogas, which is produced from a variety of organic wastes, including plant straw, through gasification (Figure 1d). Biogases include methane, hydrogen and carbon monoxide. Gasification using current technologies has a low net energy balance and its usefulness therefore might be limited [16,17]. Besides the traditional biogases, hydrogen production by green algae and microbes has been proposed as a potential source for a third generation biofuel [5]. Unlike hydrogen production from other biomass sources, algae-based hydrogen production uses a biological water-splitting reaction, in which hydrogenase uses the photosynthetic electron transport chain to reduce protons for hydrogen production [18]. Hydrogenase engineering for increased oxygen tolerance and systems biology research of genes and pathways involved in hydrogen production are needed to realize the potential of this platform [18]. Choices of crops and feedstocks Bioenergy crops can be classified into the following four groups: traditional cereal crops, traditional sugar-producing crops, dedicated lignocellulosic biomass feedstocks, and oilseed crops for biodiesel. Traditional cereal crops Cereal crops are a major source for starch-based ethanol production. Maize (Zea mays) is an important food and feed crop, used as processed food, oil, fodder, a vegetable and byproducts. Maize can be used as a bioenergy crop in two ways: the starch in seeds can be used to produce ethanol, and the crop residuals (termed stover) could potentially be used to produce lignocellulosic ethanol. Sorghum (Sorghum bicolor) is the fifth most cultivated cereal crop in the world and is grown for grain, forage, sugar and fiber. Sorghum could also be used for bioenergy in several ways. Both the starch in the grain and the sugar could be feedstock for ethanol fermentation using current technology platforms, and crop residuals could be useful for lignocellulosic ethanol production. Two features make sorghum a particularly attractive bioenergy crop. First, there would not be strong competition between the use of land for food or for energy because the seeds can be used for food and feed and the stems could be optimized for different platforms of ethanol production, which is particularly important for heavily populated developing countries such as China. Second, sorghum is drought and heat tolerant, which would enable the usage of marginal land that is not suitable for the cultivation of many other crops [19]. Besides sorghum and maize, the residuals of other crops such as wheat and rice are also expected to be useful for lignocellulosic ethanol production. Sugar-producing plants Sugar can be used for direct fermentation of ethanol. Sugarcane (Saccharum officinarum) and sugar beet (Beta vulgaris) are the major sugar-producing plants. Sugarcane is adapted to warm temperate to tropical areas, whereas sugar beet is grown in temperate areas. Therefore the two sugar crops occupy different geographical niches. Brazil is a successful example of a country that has reduced its gasoline usage by producing bioenergy. The Brazilian national ethanol program, which is based solely on sugarcane, produces 4.2 billion gallons of ethanol a year [8], although the resultant ecological and environmental effects are still debatable [20,21]. Most ethanol production using sugar beet takes place in Europe; however, using sugar beet to produce ethanol could potentially increase soil erosion and lower the net energy balance. Other sugar-producing crops include energy cane, improved cultivars of sugarcane and varieties of sweet sorghum. All the above crops are annuals, with the exception of sugarcane. Perennials are more desirable than annuals as bioenergy feedstocks because they do not need to be reseeded each growing season and therefore cultivation costs are lower. Dedicated bioenergy feedstocks Perennial bioenergy feedstocks are important sources of lignocellulosic biomass production. Switchgrass (Panicum virgatum) has been proposed as the major perennial feedstock in the USA because it is widely adapted, has high biomass production, high C-4 photosynthetic efficiency, and efficient use of water and nitrogen. Switchgrass yield is around 10 to 25 Mg/ha/yr depending on latitude, nutrition and other factors. Hybrid Miscanthus, including Miscanthus giganteus, is another highly favored biomass feedstock, mainly in Europe. However, Miscanthus giganteus is a sterile triploid clone that requires vegetative propagation. Similar to switchgrass, MisReview Trends in Plant Science Vol.13 No.8 423
Review Trends in Plant Science Vol.13 No.8 canthus x giganteus is also a C-4 perennial plant positive NEB can be considered as economically and conferring most of the advantages of switchgrass.Mis- environmentally sustainable.This is particularly import- canthus shows greater cold tolerance and hence might ant when considering which crops and conversion pro- perform better at higher latitudes.The yield of Mis- cesses might be worthy of substantial biotechnology canthus x giganteus has been reported to be between 7 investment.Even though the economics of corn starch- and 38 Mg/ha/yr and potentially has better nitrogen usage based ethanol and biodiesel production is currently com- than switchgrass [22,23]. petitive with gasoline,their NEB is fairly low or even Another group ofdedicated bioenergy feedstocks is woody negative,in contrast to the favorable NEB of lignocellu- plants,including hybrid poplar,willow and pines.Hybrid losics,as shown in Table 1 [10,28,29].If lignocellulosic poplar is considered a model woody biomass feedstock biomass can be efficiently converted into ethanol,a NEB because of its broad adaptation,available genome sequence of up to 600 GJ/ha/yr is a reasonable expectation,which and transformation techniques,and fast growth.The bio- would provide the highest NEB of all first or second mass accumulation of hybrid poplar is reported to bebe- generation platforms.Recent efforts to build biorefineries tween ~7 to 20 Mg/ha/yr depending on the nutrition and for lignocellulosic biomass processing are the first step to environmental conditions [24-26].From the perspective of fulfilling such potential;however,both low recalcitrance biomass production,switchgrass and hybrid Miscanthus feedstocks and new biocatalysts to improve the proces- seem to have the potential to produce more biomass com- sing efficiency are needed to realize this potential.Among pared with that produced by poplar.Given that a short the different bioenergy crops,switchgrass,Miscanthus, rotation for trees is five years,there is a time lag before and sorghum could potentially produce the highest NEB poplars can be harvested,and then,only the wood is har- 28.30.311 vested.Woody biomass does have a storage advantage over herbaceous feedstocks.However,geography,land-use pat- Environmental and ecological benefits of different terns,agronomy,economics and biology are likely to result platforms in multiple feedstock use.Because of the advantages of Different bioenergy platforms have different pros and cons perennial feedstock,efforts have been put into developing from an ecological and agricultural perspective(Table 1) perennial bioenergy feedstocks via breeding [27]. [41.The near-term economic advantages of ethanol pro- duction from maize and biodiesel production from soybean Plants for biodiesel are often counter-balanced by the detrimental effects of In temperate areas,annual oilseeds such as soybean agricultural practices on the environment.By contrast, (Glycine max),canola (Brassica napus),and sunfower perennial feedstocks such as switchgrass can help to (Helianthus annuus)have all been used as biodiesel feed- decrease soil erosion,improve water quality,and protect stocks.Palm oil (Arecaceae)trees have been successfully natural diversity [4,29,32-34]Perennial biomass crops used as biodiesel plants in the tropics.If we consider also complement food-based and feed-based agriculture potential biodiesel feedstocks for temperate use,the trans- instead of competing with it. portation costs for palm oil would be prohibitively expens- ive for export and would have a positive net energy Global climate change and bioenergy choices balance.In the case of soybean,canola and sunflower, Biomass crops and bioenergy production as an offset to the energy output from grain was estimated to be ~10 fossil fuel have the potential to ameliorate global warming. to 40 GJJ/ha,which is considerably lower than the ~200 Not only does the offset mean that less 'old'carbon is 500 GJ/ha energy gain from lignocellulosic biomass [28]. released into the atmosphere,but the underground bio- Hence,we might conclude that lignocellulosic biomass will mass of perennial biomass crops also acts as a carbon sink. have a greater demand than biodiesel feedstocks. For example,the capacity of Miscanthus x giganteus to fix There are other candidates for bioenergy feedstocks that carbon dioxide is estimated to be 5.2 to 7.2 t C/ha/yr,which are too numerous to detail in this review.Alternative results in a negative carbon balance where more carbon bioenergy plants include additional crops(e.g.sweet sor- dioxide is fixed than emitted [27].In a recent study of ghum),Camelina,grasses (e.g.big bluestem),trees (e.g. maize,switchgrass,soybean,alfalfa,hybrid poplar and willow),and even algae.Potentially,green algae could be reed canarygrass (Phalaris arundinacea),only poplar used for hydrogen production,oil production for biodiesel and switchgrass had a negative carbon balance(carbon platforms,and even biomass production for a bioethanol fixation of ~2t C/ha/yr)[35].However,two recent publi- platform,depending on the biotechnology breakthroughs cations indicate that first generation ethanol platforms Hydrogen is believed to be an important component of the actually have far higher carbon dioxide emissions com- third generation of bioenergy and can be adapted as differ pared with that released from fossil fuels [20,21].Accord- ent energy sources.Many factors determine the choice of ing to one of the studies,even the best lignocellulosic bioenergy crops;these are summarized in Table 1 and ethanol is predicted to have a positive carbon balance discussed further in the next section. 20].These contradictory estimates are a product of the different methods and models used to assess carbon Bioenergy:environmental,ecological and economic release and fixation.Despite the differences,there is a considerations consensus that second generation ethanol production Net energy balance of different platforms using lignocellulosic platforms should lead to a lower Net energy balance (NEB)is an important concept in carbon balance as compared with first generation plat- choosing a bioenergy platform because only a high forms.Enhancing the ability of perennial feedstocks to 424
canthus giganteus is also a C-4 perennial plant conferring most of the advantages of switchgrass. Miscanthus shows greater cold tolerance and hence might perform better at higher latitudes. The yield of Miscanthus giganteus has been reported to be between 7 and 38 Mg/ha/yr and potentially has better nitrogen usage than switchgrass [22,23]. Another group of dedicated bioenergy feedstocks is woody plants, including hybrid poplar, willow and pines. Hybrid poplar is considered a model woody biomass feedstock because of its broad adaptation, available genome sequence and transformation techniques, and fast growth. The biomass accumulation of hybrid poplar is reported to be between 7 to 20 Mg/ha/yr depending on the nutrition and environmental conditions [24–26]. From the perspective of biomass production, switchgrass and hybrid Miscanthus seem to have the potential to produce more biomass compared with that produced by poplar. Given that a short rotation for trees is five years, there is a time lag before poplars can be harvested, and then, only the wood is harvested. Woody biomass does have a storage advantage over herbaceous feedstocks. However, geography, land-use patterns, agronomy, economics and biology are likely to result in multiple feedstock use. Because of the advantages of perennial feedstock, efforts have been put into developing perennial bioenergy feedstocks via breeding [27]. Plants for biodiesel In temperate areas, annual oilseeds such as soybean (Glycine max), canola (Brassica napus), and sunflower (Helianthus annuus) have all been used as biodiesel feedstocks. Palm oil (Arecaceae) trees have been successfully used as biodiesel plants in the tropics. If we consider potential biodiesel feedstocks for temperate use, the transportation costs for palm oil would be prohibitively expensive for export and would have a positive net energy balance. In the case of soybean, canola and sunflower, the energy output from grain was estimated to be 10 to 40 GJ/ha, which is considerably lower than the 200– 500 GJ/ha energy gain from lignocellulosic biomass [28]. Hence, we might conclude that lignocellulosic biomass will have a greater demand than biodiesel feedstocks. There are other candidates for bioenergy feedstocks that are too numerous to detail in this review. Alternative bioenergy plants include additional crops (e.g. sweet sorghum), Camelina, grasses (e.g. big bluestem), trees (e.g. willow), and even algae. Potentially, green algae could be used for hydrogen production, oil production for biodiesel platforms, and even biomass production for a bioethanol platform, depending on the biotechnology breakthroughs. Hydrogen is believed to be an important component of the third generation of bioenergy and can be adapted as different energy sources. Many factors determine the choice of bioenergy crops; these are summarized in Table 1 and discussed further in the next section. Bioenergy: environmental, ecological and economic considerations Net energy balance of different platforms Net energy balance (NEB) is an important concept in choosing a bioenergy platform because only a high positive NEB can be considered as economically and environmentally sustainable. This is particularly important when considering which crops and conversion processes might be worthy of substantial biotechnology investment. Even though the economics of corn starchbased ethanol and biodiesel production is currently competitive with gasoline, their NEB is fairly low or even negative, in contrast to the favorable NEB of lignocellulosics, as shown in Table 1 [10,28,29]. If lignocellulosic biomass can be efficiently converted into ethanol, a NEB of up to 600 GJ/ha/yr is a reasonable expectation, which would provide the highest NEB of all first or second generation platforms. Recent efforts to build biorefineries for lignocellulosic biomass processing are the first step to fulfilling such potential; however, both low recalcitrance feedstocks and new biocatalysts to improve the processing efficiency are needed to realize this potential. Among the different bioenergy crops, switchgrass, Miscanthus, and sorghum could potentially produce the highest NEB [28,30,31]. Environmental and ecological benefits of different platforms Different bioenergy platforms have different pros and cons from an ecological and agricultural perspective (Table 1) [4]. The near-term economic advantages of ethanol production from maize and biodiesel production from soybean are often counter-balanced by the detrimental effects of agricultural practices on the environment. By contrast, perennial feedstocks such as switchgrass can help to decrease soil erosion, improve water quality, and protect natural diversity [4,29,32–34] Perennial biomass crops also complement food-based and feed-based agriculture instead of competing with it. Global climate change and bioenergy choices Biomass crops and bioenergy production as an offset to fossil fuel have the potential to ameliorate global warming. Not only does the offset mean that less ‘old’ carbon is released into the atmosphere, but the underground biomass of perennial biomass crops also acts as a carbon sink. For example, the capacity of Miscanthus giganteus to fix carbon dioxide is estimated to be 5.2 to 7.2 t C/ha/yr, which results in a negative carbon balance where more carbon dioxide is fixed than emitted [27]. In a recent study of maize, switchgrass, soybean, alfalfa, hybrid poplar and reed canarygrass (Phalaris arundinacea), only poplar and switchgrass had a negative carbon balance (carbon fixation of 2 t C/ha/yr) [35]. However, two recent publications indicate that first generation ethanol platforms actually have far higher carbon dioxide emissions compared with that released from fossil fuels [20,21]. According to one of the studies, even the best lignocellulosic ethanol is predicted to have a positive carbon balance [20]. These contradictory estimates are a product of the different methods and models used to assess carbon release and fixation. Despite the differences, there is a consensus that second generation ethanol production using lignocellulosic platforms should lead to a lower carbon balance as compared with first generation platforms. Enhancing the ability of perennial feedstocks to Review Trends in Plant Science Vol.13 No.8 424
Review Trends in Plant Science Vol.13 No.8 increase their below-ground biomass accumulation will be table sugars [55].Lignin biosynthesis in monocot species important for increased carbon storage [81. should be studied further so as to be able to modify lignin biosynthesis intelligently in perennial grass feedstocks Economic considerations Current biofuels were marginally profitable before recent Preprocessing in planta:expression of cellulases and petroleum prices spiked.Ethanol production from maize cellulosomes was US$0.48 per gasoline energy equivalent liter(EEL)in Plant cell walls can be degraded by individual cellulases or 2005.when the price of gasoline was US$0.46 per liter [291 in concert by cellulosomes.which consist of a suite of However,the recent spike in the price of global crude oil enzymes.In planta expression of cellulases and cellulo- has made maize-based ethanol production very profitable. somes could potentially reduce the cost of enzymatic sac- The situation is similar for biodiesel production:soybean- charification of lignocellulosic biomass at the biorefinery by based biodiesel production was US$0.55 per diesel EEL in providing the enzymes needed for cell wall degradation. 2005 [29].Despite these potential profit margins, The effectiveness of in planta expression of free cellulases traditional crop-based biofuel production has led to direct or cellulosomes is still controversial because digestion is competition between food and energy and,as a result,the complicated by the subcellular localization of enzyme(s), recent crude oil price increase has already led to the global glycosylation of extracellular proteins,effective enzyme infation of the cost of food,feed and associated products. combinations,and the requirement for inducible expres- There is an imminent need to move to lignocellulosic sion to avoid premature cell wall digestion. biomass-based platforms.However,lignocellulosic ethanol Regardless of the challenges,preliminary research has production using the current platforms is not profitable shown the successful apoplastic expression of active Acid- [361.The lignocellulosic ethanol price is still as high as othermus cellulolyticus cellulase El in maize and tobacco US$0.70 to US$1.0 per EEL.However,as processing tech- [56,57].Moreover,recent research has indicated that nologies mature and biomass crops are modified for higher maize plants showing in planta expression of cellulase yield and lower recalcitrance,the cost of lignocellulosic- had higher biomass conversion efficiency (58].Researchers based ethanol production is expected to decrease to rival have indicated that no detrimental effects were found by maize grain platforms.Indeed,based on break-even price apoplast targeting of El and that cellulase activity aided yield,recent analysis has already indicated that switch- biomass conversion when plant material was milled grass is a more profitable crop than traditional crops such [56,59].These differences result from the different sub- as sorghum and maize [10].The competitiveness of ligno- cellular localization and activation mechanisms of the cellulosic ethanol as a sustainable energy supply in the enzymes.For example,heat-activated cellulase enzymes USA will therefore heavily depend on biotechnology break- such as El have no detrimental effects to plants growing in throughs to reduce cost and improve processing efficiency. typical ambient temperatures [55.561. Further study is necessary to determine different strat- Plant biotechnology solutions for bioenergy egies for in planta cell wall digestion enzyme expression, Novel enabling biotechnologies are crucial for reducing the with high-throughput approaches for optimized sub-cellu- costs of bioenergy production,particularly of lignocellulo- lar localization and different combinations of enzymes.In sic ethanol.The key issues include rapid domestication, addition,fundamental research still needs to be performed overcoming recalcitrance,efficient breakdown of cellulose, to modify cellulases for improved catalytic efficiency,ther- and increasing biomass and lipid production for ethanol mal stability,performance under extreme conditions, and biodiesel,respectively [37].Although aspects of these along with protein modifications designed to lead to important areas will be discussed individually below,it is reduced apoplastic glycosylation.Many cell-wall degrading important to solve these problems in concert. bacteria use cellulosomes,a suite of enzymes for cell wall hydrolysis [60].Cellulosomes have not yet been expressed Modification of lignin biosynthesis in transgenic plants,but the correct assemblage of cellulo- Lignin might be the most crucial molecule in need of some components in the plant apoplast potentially have modification for lignocellulosic feedstocks.It has been the promise of decreasing recalcitrance and facilitating the established that reducing lignin biosynthesis can lead to post-harvest hydrolysis of cellulose,which in turn might lower recalcitrance and higher saccharification efficiency aid simultaneous saccharification and fermentation [13.38-54].Recent studies have indicated two important aspects for lignin modification.First,both lignin content Abiotic stress resistance and composition are important.Although it is codependent Suboptimal water and other abiotic stresses are limiting on efficient processes to fractionate lignin,a more uniform factors for biomass production;stress tolerance traits are lignin structure might facilitate more efficient cell-wall therefore important to enable feedstock to be produced on degradation for fuel production (John Ralph,personal marginal or sub-marginal lands not favorable for food communication).Second,the pretreatment of biomass crops.Drought-,metal-,salt-,cold-and heat-stress all might even be rendered unnecessary if lignin content falls induce some similar responses in plants,yet each of these below a critical threshold,which would enhance down- stresses will induce a different set of genes [61].The stream enzymatic saccharification and fermentation steps upstream pathways for salt and drought stresses have for improved efficiency [55].Therefore,switchgrass,Mis- been well-characterized in Arabidopsis thaliana [62-64], canthus or poplar feedstocks with modified lignin can but until recently have led to only limited success in improve the efficiency of biomass conversion into fermen- translational research to produce field crop abiotic stress 425
increase their below-ground biomass accumulation will be important for increased carbon storage [8]. Economic considerations Current biofuels were marginally profitable before recent petroleum prices spiked. Ethanol production from maize was US$0.48 per gasoline energy equivalent liter (EEL) in 2005, when the price of gasoline was US$0.46 per liter [29]. However, the recent spike in the price of global crude oil has made maize-based ethanol production very profitable. The situation is similar for biodiesel production: soybeanbased biodiesel production was US$0.55 per diesel EEL in 2005 [29]. Despite these potential profit margins, traditional crop-based biofuel production has led to direct competition between food and energy and, as a result, the recent crude oil price increase has already led to the global inflation of the cost of food, feed and associated products. There is an imminent need to move to lignocellulosic biomass-based platforms. However, lignocellulosic ethanol production using the current platforms is not profitable [36]. The lignocellulosic ethanol price is still as high as US$0.70 to US$1.0 per EEL. However, as processing technologies mature and biomass crops are modified for higher yield and lower recalcitrance, the cost of lignocellulosicbased ethanol production is expected to decrease to rival maize grain platforms. Indeed, based on break-even price yield, recent analysis has already indicated that switchgrass is a more profitable crop than traditional crops such as sorghum and maize [10]. The competitiveness of lignocellulosic ethanol as a sustainable energy supply in the USA will therefore heavily depend on biotechnology breakthroughs to reduce cost and improve processing efficiency. Plant biotechnology solutions for bioenergy Novel enabling biotechnologies are crucial for reducing the costs of bioenergy production, particularly of lignocellulosic ethanol. The key issues include rapid domestication, overcoming recalcitrance, efficient breakdown of cellulose, and increasing biomass and lipid production for ethanol and biodiesel, respectively [37]. Although aspects of these important areas will be discussed individually below, it is important to solve these problems in concert. Modification of lignin biosynthesis Lignin might be the most crucial molecule in need of modification for lignocellulosic feedstocks. It has been established that reducing lignin biosynthesis can lead to lower recalcitrance and higher saccharification efficiency [13,38–54]. Recent studies have indicated two important aspects for lignin modification. First, both lignin content and composition are important. Although it is codependent on efficient processes to fractionate lignin, a more uniform lignin structure might facilitate more efficient cell-wall degradation for fuel production (John Ralph, personal communication). Second, the pretreatment of biomass might even be rendered unnecessary if lignin content falls below a critical threshold, which would enhance downstream enzymatic saccharification and fermentation steps for improved efficiency [55]. Therefore, switchgrass, Miscanthus or poplar feedstocks with modified lignin can improve the efficiency of biomass conversion into fermentable sugars [55]. Lignin biosynthesis in monocot species should be studied further so as to be able to modify lignin biosynthesis intelligently in perennial grass feedstocks. Preprocessing in planta: expression of cellulases and cellulosomes Plant cell walls can be degraded by individual cellulases or in concert by cellulosomes, which consist of a suite of enzymes. In planta expression of cellulases and cellulosomes could potentially reduce the cost of enzymatic saccharification of lignocellulosic biomass at the biorefinery by providing the enzymes needed for cell wall degradation. The effectiveness of in planta expression of free cellulases or cellulosomes is still controversial because digestion is complicated by the subcellular localization of enzyme(s), glycosylation of extracellular proteins, effective enzyme combinations, and the requirement for inducible expression to avoid premature cell wall digestion. Regardless of the challenges, preliminary research has shown the successful apoplastic expression of active Acidothermus cellulolyticus cellulase E1 in maize and tobacco [56,57]. Moreover, recent research has indicated that maize plants showing in planta expression of cellulase had higher biomass conversion efficiency [58]. Researchers have indicated that no detrimental effects were found by apoplast targeting of E1 and that cellulase activity aided biomass conversion when plant material was milled [56,59]. These differences result from the different subcellular localization and activation mechanisms of the enzymes. For example, heat-activated cellulase enzymes such as E1 have no detrimental effects to plants growing in typical ambient temperatures [55,56]. Further study is necessary to determine different strategies for in planta cell wall digestion enzyme expression, with high-throughput approaches for optimized sub-cellular localization and different combinations of enzymes. In addition, fundamental research still needs to be performed to modify cellulases for improved catalytic efficiency, thermal stability, performance under extreme conditions, along with protein modifications designed to lead to reduced apoplastic glycosylation. Many cell-wall degrading bacteria use cellulosomes, a suite of enzymes for cell wall hydrolysis [60]. Cellulosomes have not yet been expressed in transgenic plants, but the correct assemblage of cellulosome components in the plant apoplast potentially have the promise of decreasing recalcitrance and facilitating the post-harvest hydrolysis of cellulose, which in turn might aid simultaneous saccharification and fermentation. Abiotic stress resistance Suboptimal water and other abiotic stresses are limiting factors for biomass production; stress tolerance traits are therefore important to enable feedstock to be produced on marginal or sub-marginal lands not favorable for food crops. Drought-, metal-, salt-, cold- and heat-stress all induce some similar responses in plants, yet each of these stresses will induce a different set of genes [61]. The upstream pathways for salt and drought stresses have been well-characterized in Arabidopsis thaliana [62–64], but until recently have led to only limited success in translational research to produce field crop abiotic stress Review Trends in Plant Science Vol.13 No.8 425