Various processes generating transport biofuels start from complex organic feed­stocks, including complete organisms or large chunks thereof, or from wastes such as sewage sludge, black liquor or household waste. The focus of such processes is often on lignocellulose. This is not surprising as the share of lignocellulose in all biomass has been estimated at about 50% (Claassen et al. 1999). Lignocellulose is a structural material of plants and a composite of lignin (a polymer composed of monolignols), cellulose (a glucose polymer) and hemicellulose (a polymer made up of diverse hexose and pentose sugars). The US Energy Law of 2008 stipulates that from 2016, transport bioethanol producers must switch to lignocellulosic feed­stocks.

There are a wide variety of lignocellulosic feedstocks. Wood, wood waste, har­vest residues, a variety of wastes and by-products originating in industries are rela­tively rich in lignocellulose (Prasad et al. 2007). It also has been suggested that nat­ural grasslands can be exploited as a source for lignocellulosic feedstock (Tilman et al. 2006; Zhou et al. 2008). It is furthermore possible to grow lignocellulosic crops on plantations. Examples of species that are considered for this purpose are woody perennials, such as eucalyptus, poplar, willow and black locust, and grasses and other non-woody perennials, such as switchgrass, elephant grass, reed canary grass, Miscanthus, cardoon, reeds and Bermuda grass. Of these species, Bermuda grass and reed canary grass are currently used as forage for livestock (Boateng et al. 2007; Pahkala et al. 2008).

The relative amount of lignin in lignocellulose is source dependent. In nutshells, the percentage of lignin may be 30-40% and in rice straw about 5.5% (Prasad et al.

2007) . Similarly, the composition of hemicellulose is source dependent. For in­stance, hemicellulose from agricultural residues or hardwood tends to be rich in pentose sugars, whereas such sugars are a minor component in hemicellulose from softwood (Hahn-Hagerdal et al. 2007).

Apart from lignocellulose, the lignocellulosic feedstocks also contain a variety of other compounds, both organic and inorganic in character. The latter, a for­tiori, holds for complex wastes such as sludges from wastewater treatment plants or household wastes, which have been proposed as sources of transport biofuel (Ptasin — ski et al. 2002). There is also a relatively limited supply of cellulosic wastes that may be used for biofuel production, such as sludges from (virgin) paper produc­tion and paper recycling (Mabee and Roy 2003; Prasad et al. 2007; Marques et al.

2008) .

There are several ways to generate substances that may serve as automotive bio­fuels from complex organic feedstocks. A first possibility, which applies both to biomass in general and to lignocellulose, is heating (‘thermochemical treatment’) to produce liquid biofuels. An option which has been exploited for centuries is the dry distillation or slow pyrolysis of wood. Apart from charcoal, methanol is an output (approximately 1-2% by weight) of slow pyrolysis of wood, which can in principle be used as a transport fuel (Reinharz 1985; Demirba§ 2001; Gullu and Demirba§ 2001; Huber et al. 2006). More recently, much attention has been given to fast and flash pyrolysis of biomass (Goyal et al. 2008). Fast and flash pyrolysis of biomass in principle produces charcoal or biochar, gas, organic fluids and water. The precise nature of the products and the relative shares of the different components can be varied, dependent on the character of the biomass, the presence of inorganic sub­stances (especially metals), reactor design, temperature, heating rate, catalysts and reaction time (Bridgwater et al. 1999; Yang et al. 2004; Demiral and §ensoz 2006; Huber et al. 2006; Boateng et al. 2007; Dobele et al. 2007; Lange 2007; Muller — Hagedorn and Bockhorn 2007; Demirba§ 2008; Di Blasi 2008; Fahmi et al. 2008; Ros et al. 2009). The fluid produced by fast and flash pyrolysis contains water and a variety of organic compounds, the latter collectively called ‘pyrolysis oil’. The pyrolysis oil tends to be unstable and to show polymerization reactions (Fahmi et al. 2008). It needs upgrading to serve as a basis for transportation fuel, for example, by hydrodeoxygenation, hydrogenation or treatment with zeolites (Huber et al. 2006; Esler 2007; Wang et al. 2008b). Such upgrading has proven difficult, and this has re­stricted the application of biomass pyrolysis technology (Wang et al. 2008b). It has also been proposed to view the pyrolysis oil as a basis for a biorefinery generating a number of chemicals besides transport fuel (Hayes 2008). As an alternative to fast and flash pyrolysis, a process has been proposed that combines pyrolysis with hy — drogenationby a formic acid-alcohol mixture (Kleinert and Barth 2008). Also under development is deoxy-liquefaction (Goyal et al. 2008; Wang et al. 2008), converting lignocellulosic biomass into a liquid that tends to be richer in hydrocarbons than the liquids commonly produced by fast pyrolysis.

A second possibility to deal with complex organic feedstocks is based on gasifi­cation of biomass or lignocellulosic materials resulting in the formation of synthesis gas (containing relatively high percentages of CO and H2). The formation of tar, and to a lesser extent char, and ash-related problems have emerged as problems in such gasification, necessitating major efforts in the field of optimizing gasification, tar reforming and syngas quality control and clean-up (Wang et al. 2008b). Using the water shift reaction, the amount of H2 in synthesis gas may be maximized, and H2 and the other main product of the water shift reaction (CO2) can be separated by processes such as pressure swing adsorption, membrane separation and cryogenic separation (Ferreira-Aparicio et al. 2005; Andersson and Harvey 2006; Haryanto et al. 2007; Barelli et al. 2008; Florin and Harris 2008; Wang et al. 2008b). It is also possible to subject syngas (after clean-up) to catalytic methanation, generating synthetic natural gas (Felder and Dones 2007).

Alternatively, conversion is possible into liquids (biomass-to-liquids or BTL biofuels). One option is the use of synthesis gas to produce oxygenates such as methanol (Reed and Lerner 1973; Demirba§ 2001; Ptasinski et al. 2002) and dimethylether (Joelsson and Gustavsson 2008). Producing ethanol from syngas is also possible but is as yet not very efficient (Subramani and Gangwal 2008). Still another option is to use the Fischer-Tropsch reaction, after enrichment of syngas with hydrogen, to generate hydrocarbons (Dietenberger and Anderson 2007), or the methanol-to-synfuel synthesis to produce hydrocarbons (Takeshita and Yamaji 2008). The latter can be conveniently applied in diesel or Otto motors (Reinhardt et al. 2006) or in airplanes (Esler 2007). There are also bacteria that can convert synthesis gas into ethanol, and these are currently researched for use in biofuel production (Henstra et al. 2007; Tollefson 2008). Low conversion rates, product inhibition and problems in maintaining optimum conditions have for a substantial time prevented commercialization of this approach (Wang et al. 2008b), but such problems have now apparently been solved to the extent that a pilot plant has been announced (Ashley 2008).

Thirdly, cellulose and hemicellulose present in lignocellulose may be enzymat­ically converted into ethanol or butanol, to be applied in, for example, Otto mo­tors (Sanchez and Cardona 2008; Qureshi et al. 2008a, b). This requires separating hemicellulose from lignin, hydrolysis of cellulose and hemicellulose into sugars and fermentation of the sugars generated by hydrolysis (Lynd 1996; Lachke 2002; Palmarola-Adrados et al. 2005; Gray et al. 2006; Angenent 2007; Prasad et al. 2007; Gomez et al. 2008; Sanchez and Cardona 2008; Qureshi et al. 2008a, b).

Hydrolysis of cellulose generates glucose, which can be converted into ethanol. Important among the hydrolytic products of hemicellulose is often xylose, a 5-car­bon sugar (Fortman et al. 2008). Xylose can be converted into ethanol by fermenta­tion as follows:

3D-xylose (C5H10O5) ^ 5ethanol + 5CO2 .

Micro-organisms such as Pichia stipitis and genetically modified Escherichia coli are able to perform the fermentation of xylose (Rubin 2008). Minor sugars originat­ing in cellulose and hemicellulose are arabinose, rhamnose, glucose, galactose and mannose, which can be converted into ethanol, too (Numan and Bhosle 2006; Fort — man et al. 2008; Hayes 2008). It is also possible to ferment C6 and C5 sugars into a mixture of acetone, butanol and ethanol (Jones and Woods 1986; Qureshi et al. 2008c). Process design tends to be focused on a limited number of lignocellulosic feedstocks for which the process is optimized (Olofsson et al. 2008). In practice, the separation of hemicellulose from lignin currently causes most problems, which are in part linked to the heterogeneous structure of lignin polymers (Gomez et al. 2008; Wackett 2008). Building cell walls involves many enzymes (McCann and Carpita 2008), and it may well be that a combination of enzymes may be necessary for their deconstruction in a way that is optimal for the next step of biofuel production: sac­charification. However, most processes currently studied for near-term application rely on the use of rather brute physico-chemical force to separate the constituents of lignocellulose (which negatively impacts overall energy efficiency and the envi­ronmental burden). Examples are: the use of acid (whether or not combined with ionic liquid), steam explosion (sometimes combined with oxidation), high-pressure hot water treatment, treatment with alkaline peroxides and ammonia fibre explosion (Huber et al. 2006; Gomez et al. 2008; Li et al. 2008; S0rensen et al. 2008; Qureshi et al. 2008c). The difficulty of separation varies for different plant species (Bura­nov and Mazza 2008). Coniferyl lignin appears, for instance, more recalcitrant so far against physico-chemical methods of separation than syringyl lignin (Anderson and Akin 2008). And the presence of oxidatively coupled esterified or etherified fer- ulic acid residues has also been reported to inhibit separation (McCann and Carpita 2008).

Proposals to overcome the hurdles to separation of lignin and cellulose and hemicellulose include: application of lignin-degrading white rot fungi of micro­organisms derived from termite guts, of Clostridium phytofermentans, and pre­treatment with phenolic esterases (Warnick et al. 2002; Anderson and Akin 2008; Rotman 2008; Weng et al. 2008). Also, it has been suggested to use lignases and to convert degraded lignin into transport biofuels (Blanch et al. 2008). Furthermore, there have been proposals to downregulate lignin biosynthesis in plants by genetic modification to ease the release of cellulose and hemicellulose and ultimately sug­ars from plants (Chapple et al. 2007; Wackett 2008). Such downregulationhas led to plant characteristics that are unsuitable for biofuel crops, such as increased suscep­tibility to fungi, dwarfing and the collapse of vessels in xylem (Weng et al. 2008). Dwarfing has been linked to the simultaneous inhibition of flavonoid production (McCann and Carpita 2008). There have been new proposals for genetic modifica­tion, focusing on changes in lignin polymer structure and monolignol polymeriza­tion (Weng et al. 2008), but it is as yet not clear whether this approach will lead to suitable biofuel feedstocks.

Degradation of hemicellulose may also be difficult. Hemicelluloses appear so far refractory against saccharification when esterified by ferulic or coumaric acids (Anderson and Akin 2008). And enzymatic hydrolysis of cellulose to fermentable sugars currently requires, per kg ethanol produced, 40-100 times more enzyme than the hydrolysis of starch (Eijsink et al. 2008). This has led to the proposal to include glycosyl hydrolases into plants by genetic modification (Taylor et al. 2008). Hy­drolysis and fermentation of cellulose and hemicellulose can be done in a two-step process (e. g. Zanichelli et al. 2007; Hayes 2008), with one hydrolytic and one fer­mentative step. When dilute acid is used for the pre-treatment of lignocellulosic biomass, there is often much hydrolysis of cellulose and hemicellulose. At higher temperatures, dilute acid treatment may also lead to much hydrolysis of cellulose (Hayes 2008). A variant of treatment with dilute acid may be used to generate sub­stantial amounts of the platform chemicals furfural and levulinic acid, in line with biorefinery concepts (Hayes 2008). Alternatively, hydrolytic enzymes produced by micro-organisms may be used (Lynd et al. 2002; Demain et al. 2005; Desvaux 2005).

The two steps in the conversion of cellulose and hemicellulose to ethanol may also be combined in a one-step process: simultaneous saccharification and fermen­tation (SSF). Simultaneous saccharification (hydrolysis of cellulose and hemicellu­lose giving rise to sugars) and fermentation by micro-organisms is often preferred as it is associated with shorter residence times and potentially higher yields and lower costs (Ballesteros et al. 2004; Demain etal. 2005; Huber etal. 2006; Angenent 2007; Marques et al. 2008). In simultaneous saccharification and fermentation, sacchari­fication is the rate-limiting step. Inhibition of fermentation by substances formed during pre-treatment and hydrolysis is a problem. Inhibitory compounds formed during pre-treatment and hydrolysis include salts, phenols, furfural, cinnamalde — hyde, ^-hydroxybenzaldehyde, lignin monomers and syringaldehyde (Zanichelli et al. 2007; Qureshi et al. 2008c; Sanchez and Cardona 2008; Royal Society 2008). The presence of inhibitors often necessitates the ‘detoxification’ by physical, chem­ical or biological methods. Another option is the use of fermenting organisms that are more tolerant to inhibitors (Hayes 2008; Olofsson et al. 2008).

Processes converting cellulose and hemicellulose into ethanol have as yet rel­atively low sugar-to-ethanol efficiencies, if compared with the well-established starch — or sucrose-to-ethanol conversion processes (Chang 2007; Hahn-Hagerdal et al. 2007; Olofsson et al. 2008). In view of the problems in converting ligno — cellulosic feedstocks into alcohol, there is a lively search for improvements, if not a ‘technological breakthrough’ or a ‘superbug’ that is able to perform the task of converting lignocellulose into ethanol with sufficient efficiency (Eijsink et al. 2008; Gomez et al. 2008; Rotman 2008).

In North America and Scandinavia, ethanol from woody lignocellulose has been, and still is, produced as a by-product of sulphite pulping for the paper industry (McElroy 2007). Wood hydrolysate is in this case converted into ethanol by yeast — based fermentation. Low nutrient concentrations, a large proportion of xylose and the presence of fermentation inhibitors have limited the efficiency thereof, and there are proposals for the optimization of sulphite liquor fermentation (Helle et al. 2008).

In Russia, there is a long-standing, large-scale, yeast-based production of ethanol from sugars obtained from wood chips hydrolyzed at elevated temperature by treat­ment with concentrated sulphuric acid (Bungay 2004; Zverlov et al. 2006). In Brazil, Europe and the USA, there are pilot plants producing ethanol from lignocellulose or components thereof such as cellulose and hemicellulose (Wheals et al. 1999; Bryner 2007a). Large-scale plants are under construction and consideration. In part, ethanol production from lignocellulose in such plants is combined with ethanol production based on sugar or starch.

Alternatively, a bacterial fermentation process for the production of the biofuel butanol from lignocellulose may be considered (Zverlov et al. 2006; Ezeji et al. 2007; Qureshi et al. 2008a, b). This process was used during the twentieth cen­tury in the Soviet Union for the fermentation of hydrolyzed lignocellulosic wastes (Zverlov et al. 2006). In this case, lignocellulose was hydrolyzed by treatment with high concentrations of sulphuric acid, and the hydrolysate was fermented in combi­nation with the fermentation of starch. H2 originated as a by-product (Zverlov et al.

2006) . Also, processes producing butanol from lignocellulose based on treatment with dilute acid followed by enzymatic treatment and fermentation (simultaneous saccharification and fermentation) of harvest residues have been proposed (Ezeji et al. 2007; Qureshi et al. 2008a, b), as have been processes to convert complex or­ganic feedstocks into mixtures of alcohols using mixtures of fermentative bacteria (Bagajewicz et al. 2007). Finally, there is research into the possibility of enzymati­cally converting lignocellulose into fatty acid ethyl esters (Royal Society 2008).

Fourthly, methane may be produced from complex organic materials. Methane is also the molecule that makes natural gas a fuel, and natural gas supplies currently about 3% of primary energy for transport (de la Rue du Can and Price 2008). In 2004, there were about 3 million motorcars powered by natural gas, usually biva­lent vehicles able to drive on compressed natural gas and gasoline (Dondero and Goldemberg 2005; Janssen et al. 2006). Substantial use of vehicles powered by nat­ural gas is found in Argentina (world leader with about 800,000 of such vehicles by

2005) , India, Pakistan, Brazil, the USA and some countries in the European Union, such as Italy (Janssen et al. 2006). Large-scale application of methane in cars is dependent on a good refuelling infrastructure (Janssen et al. 2006). Natural gas is also used in ship and on-farm transport (Royal Society 2008; Boijesson and Mat — tiasson 2008). Alternatively, methane may be converted into liquid fuels using the Fischer-Tropsch reaction or via a process with ethylene as an intermediary (Hall

2005) . Currently, use of methane from natural gas in the Fischer-Tropsch synthesis of hydrocarbons is applied in diesel production, and this application is expected to increase in the future (Bagajewicz et al. 2007; Bryner 2007b; Takeshita and Yamaji 2008). Methane can furthermore be converted into methanol (Huber et al. 2006; Cantrell et al. 2008).

The use of methane in transport and the production of other transport fuels may be extended to biogenic methane (Murphy and McCarthy 2005; Boijesson and Mattiasson 2008; Lehtomaki et al. 2008). Above, the production of synthetic nat­ural gas from syngas has already been referred to. Methane can also be produced from a wide variety of biomass and biomass-derived materials, including complex wastes, using mixed cultures of micro-organisms in anaerobic reactors (Murphy and McCarthy 2005; Kleerebezem and van Loosdrecht 2007; Bocher et al. 2008; Ros et al. 2009). It has been proposed to use marginal lands for the large-scale growth of feedstocks and convert those into methane in decentralized biogas reac­tors (Schroder et al. 2008). A variant of this approach has been suggested that also allows for the bioconversion of CO2 to CH4 (Alimahmoodi and Mulligan 2008). Landfills can also be exploited for methane production. Before application in trans­port, CH4 production from biomass should be followed by upgrading. The extent of upgrading necessary varies, depending on the methane source. More upgrading is usually needed for methane from refuse in landfills and sewage sludge than for methane from manure (Rasi et al. 2007). Upgrading partly serves to remove com­pounds that may negatively affect engine performance or emissions, such as halo — genated compounds, siloxanes, H2S and NH3 (Ferreira-Aparicio et al. 2005; Rasi et al. 2007). Upgrading may also aim to increase methane content.

Hydrocarbons (‘Biocrude’) from Terrestrial Plants

In the 1930s, there were some efforts to cultivate Euphorbia, producing hydrocar­bons for biofuel production (Kalita 2008). Subsequently, there has been substantial research regarding plants producing latex which may be cracked to yield transport biofuels. The Euphorbia lathyris did relatively well in this respect and has been calculated to yield about 48 MJ ha-1 year-1 in biofuel: 26 MJ as hydrocarbons and 22 MJ as ethanol (Kalita 2008). As will be shown in Chap. 2, such energetic yields are relatively low compared with biofuels from current terrestrial crops, and there is no current commercial application of ‘biocrude’ from terrestrial plants.