The polymer was discovered by (Shima and Sakai, 1977). The polymer has been found to be heat stable, and can even withstand autoclaving for 20 min. These properties led to the use of polylysine as a food preser­vative on a commercial scale in Japan (Yoshida and Nagasawa, 2003). Production of the compound is influ­enced substantially by the pH of the medium. Polylysine has not known to form any secondary or tertiary struc­ture, and its microbicidal activity is attributed to its pol­ycationic nature.

Production of e-Poly-L-Lysine

The strain Streptomyces albulus 346 spp. lysinopolyme — rus was the first organism to be isolated as a polylysine producer, following which many improvements were carried out on the same for industrial production of polylysine. Later more producers from the genus Streptomyces, Kitasatospora, and an ergot fungus epichole were found to produce polylysine. Polylysine is now industrially produced by aerobic fermentation, us­ing a mutant derived from S. albulus 346, isolated from soil. A maximum amount of polylysine, 0.5 g/l was reported under optimized conditions set at pH 6.0 (Shima and Sakai, 1981). A mutant strain resistant to S-(2-aminoethyl)-L-cysteine, an analogue for lysine and glycine were derived and gave higher productivity values of up to 20 mg/l of polylysine, after 120 h, with glucose as the carbon source and ammonium sulfate as the nitrogen source (Hiraki et al., 1998).

Streptomyces albulus 410, the strain that has been exploited for commercial production of polylysine displayed the two-stage polylysine production (pH 6.0 and pH 3.0—5.0). Accurate control of the process and pH led to a maximal production of about 48.3 g/l. It was found that the best pH for increasing the cellular mass was pH 6.0, while pH below 4.2 was favorable for high levels of polylysine production (Shi et al., 2007). Every poly(amino acid) produced is poly dispersed (has variable molecular weight), which in turn makes it difficult to obtain the product of the required specification. This problem was recently tackled to an extent, when new isolates belonging to the genus Streptomyces was obtained, which could pro­duce nearly monodispersed polylysine.

Degradation of Polylysine

There is not much report on the biodegradation of polylysine. The polylysine-resistant strain Chryseobacte — rium sp. OJ7 also was postulated to have a polylysine­degrading enzyme, with an exopeptidase activity (Obst and Steinbuchel, 2004). Polylysine was shown to be susceptible and was degraded by commercially avail­able enzymes proteases A, P and peptidase R from A. oryzae, Aspergillus melleus and Rhizopus oryzae (Kito et al., 2002).

Applications of Polylysine

The application of polylysine as a food preservative has been established. Other uses of polylysine include the production of an emulsifying agent through its conjugation with dextran. Polylysine can be used to coat biochips and surfaces of cell culture flasks, so as to provide a biocompatible adherent surface. One of the most industrially relevant applications of polylysine is its use as a drug delivery agent and an aid in cell trans­formation due to its polycationic nature.

Hydrogen

Frequently cited as the fuel of the future, hydrogen production, storage and utilization are being widely investigated. As a transportation fuel it presents a series of challenges in every link of the chain, from production to storage and distribution. Although having a low volu­metric energy density, hydrogen has the highest energy density per mass and the simple fact that its combustion generates almost only water and heat has seduced entire generations. "Yes, my friends, I believe that water will one day be employed as fuel, that hydrogen and oxygen which constitute it, used singly or together, will furnish an inexhaustible source of heat and light, of an intensity of which coal is not capable" (Verne). Cars that could run on water with minimal energy consumption have captured the imagination of many people and, not sur­prisingly, have inspired frauds like the almost magical conversion of saltwater into fuel using radiofrequency radiation, claimed by John Kanzius and broadcast live countrywide from Philadelphia, or the notorious "Stan — leyMeyer’s water fuel cell" to be used in an internal combustion engine, where a special device could split water giving an energy output sufficient to generate me­chanical energy for the vehicle with enough leftover to power a fuel cell that would provide more hydrogen and oxygen through water splitting. Considering that the combustion of hydrogen and oxygen regenerates water, both systems obviously defy the first and second laws of thermodynamics (Ball, 2007).

Despite the motivation behind these schemes, they touched upon the most limiting step in the development of the hydrogen fuel technology: production. In current industrial practice, hydrogen can be produced by pyrol­ysis, electrolysis or by steam reforming of hydrocarbons. The last is the dominant method, applied to fossil fuels, usually natural gas (methane). This makes hydrogen both expensive and unsustainable.

Hydrogen Bioproduction

Molecular hydrogen (H2) is the lightest gas possible. When released into the atmosphere it diffuses quickly toward the troposphere, thus, at the sea level it can only be found in trace amounts. For this reason, very lit­tle naturally occurring H2 is available and therefore a sustainable production system must be found if this molecule is to be used as a fuel. Efficient biological production of hydrogen could represent a breakthrough in the development of this energy carrier and many different approaches are being followed toward this goal. Undoubtedly, among all the possible fuels that could be produced by cyanobacteria, it is hydrogen that has received the most attention. Here we discuss the biological mechanisms for hydrogen production and advances toward yield improvements in cyanobacteria.

In the light reactions of photosynthesis, light is captured by photosystems I and II, acting together to transform solar energy into chemical energy, splitting water into molecular oxygen and protons (H+) and the reducing agent NADPH. The transmembrane proton gradient that is formed is used by ATP synthase to combine adenosine diphosphate (ADP) + Pi into ATP (Figure 22.1). This set of reactions is rather interesting because it effectively conserves ubiquitous solar energy in energy-dense molecules using an abundant substrate, water. Ironically, cyanobacteria (and all plants) had been all along for millions of years the very sought after solu­tion for breaking the strong bond between oxygen and hydrogen in the water molecule without using the spe­cial radiofrequency of John Kanzius or the mysterious fuel cell of Stanley Meyer.

During the water-splitting process, oxygen is released in its molecular form (O2), while hydrogen, in the form of protons, is further used to produce two molecules of high-energy content: ATP and NADPH. Together, they feed energy into the Calvin-Benson-Bassham cycle, where CO2 is fixed into organic molecules, as well as into many other reactions related to cellular homeostasis or secondary metabolism. Alternatively, before it is used to generate NADPH, the high-energy electron generated by photosynthesis can be directly used for the evolution of hydrogen, a process called direct bio­photolysis (Benemann and Weare, 1974). Therefore, hydrogen evolution through this route does not require CO2 fixation, and solar energy and water, together with the required enzymes, are sufficient for H2 formation (Hallenbeck and Benemann, 2002). The major problem with this process is that hydrogenases, the hydrogen — evolving enzymes, are extremely sensitive to oxygen (O2) and are irreversibly inactivated by even small concentrations of this gas. Thus, hydrogen evolution is usually a short-lived process, with a burst of hydrogen evolution when transitioning from a dark cycle into light as increasing oxygenic photosynthesis quickly inactivates the hydrogenase. Some species, especially filamentous ones (e. g. Anabaena sp. and Nostoc sp.), capable of forming specialized cells called heterocysts, can be shown to produce hydrogen over prolonged periods in light, as the heterocysts provide an oxygen-free environment that protects the hydrogenase against inactivation. In indirect biophotolysis, the captured light energy is used to fix CO2 and the organic molecules that are pro­duced are stored as reserve material. Under normal conditions, part of these carbon reserves will be oxidized over the dark period to maintain cellular ho­meostasis. However, under proper conditions such a culture can be induced to produce hydrogen, thus separating hydrogen evolution temporally and spatially from the oxygen evolved by oxygenic photo­synthesis (Hallenbeck, 2011). Thus, hydrogenase activ­ity is maintained and the simultaneous production of hydrogen and oxygen, an explosive mixture when concentrated in the headspace of a bioreactor, is avoided.

Current bioenergy resources consist of residues from forestry and agriculture, various organic waste streams and dedicated biomass production from pasture land, wood plantations and sugar cane (Figure 2.2). At pre­sent, the main biomass feedstocks for electricity and heat generation are forestry and agricultural residues and municipal waste in cogeneration and cofiring power plants. In the longer term, lignocellulosic crops could provide bioenergy resources for second-generation bio­fuels, which are considered more sustainable, provide land use opportunities and will reduce the competition with food crops (http://www. ga. gov. au/image_cache/ GA16706.pdf).

Major feedstock sources for future biofuel production are likely to be high biomass producing plant species such as poplar, pine, switchgrass, sorghum maize, Miscanthus, hemp, Jatropha, willow and cassava. With
growing interest in the utilization of plant biomass for the production of ethanol and other biofuels, the use of plant species as biofuel feedstocks has become a focal point in research. Due to concerns about diverting grain and seed from human food and livestock feed to biofuel feedstock production, emphasis has shifted to the use of lignocellulose-derived biofuel production, and research is now directed at improving not only lignocellulosic yield but also quality traits in these species (Banerjee, 2011; Mueller et al., 2011; Tyner, 2010).

A long-term opportunity exists to produce fuels from nonedible lignocellulosic biomass from plants (Heather and Somerville, 2012). Sugarcane, energy cane, elephant grass, switchgrass, and Miscanthus have intrinsically higher light, water and nitrogen use efficiency and are fast-growing biomass/crops for bioenergy work pro­gram. Work on perennial grasses such as switchgrass (Panicum spp.), prairie cordgrass (Spartina spp.), big bluestem (Andropogon spp.), little bluestem (Schizachy — rium spp.) and others could produce significant biomass in a variety of biomass throughout the northern plains and southeastern grasslands in the United States (Gonzalez-Hernandez et al., 2009). Woody biomass can be harvested sustainably for lumber and paper and may, therefore, provide biofuel feedstock for some re­gions (Malmsheimer et al., 2011). Table 2.5 summarizes the countrywise contribution of current biofuel yield from different feedstocks.

As mentioned previously, biomass energy can come from numerous sources and produce several types of fuels. Ethanol is typically produced from biomass

Wood industry

residues 5%

Recovered

wood 6%

Municipal solid

waste and landfill gas

high in carbohydrates (sugar, starch and cellulose) dur­ing a fermentation process. Recent developments in fermentation processes now allow almost any plant type to be used to produce ethanol. The most prom­ising natural oils, such as rapeseed oil, have been used to produce biodiesel, which performs much like petroleum-derived diesel fuel. Apart from agricultural, forestry and other by-products, the main source of lignocellulosic biomass for second-generation biofuels is likely to be from "dedicated biomass feedstocks", such as certain perennial grass and forest tree species. Genomics, genetic modifications and other biotechnol­ogies are all being investigated as tools to produce plants with desirable characteristics for second — generation biofuel production, for example, plants that produce less lignin (a compound that cannot be fermented into liquid biofuel), plants that produce en­zymes themselves for cellulose and/or lignin degrada­tion, or plants that produce increased cellulose or overall biomass yields. Grass, leaves, agri crops, agri­crop residues and currently available nonfood plant biomass are the dominant source of lignocellulosic materials (Carpita, 2012; Ambavaram et al., 2011;

Abramson et al., 2010; Davison et al., 2006; Nguyen et al., 1999, 2000).

Bioenergy resources used in current biofuels develop­ment programs, potential future resources and the related bioenergy outputs are summarized in Table 2.6. Bioenergy resources are difficult to estimate due to their multiple and competing uses. Production statistics exist for current commodities such as grain, sugar, pulp wood and saw logs; however, these commodities are currently largely committed to food, animal feed and materials markets. Potential feedstocks for the future include modified strains of existing crops, new tree crops and algae. There are many factors to be taken into ac­count for each bioenergy resource, such as moisture con­tent, resource location and distribution, and type of conversion process that is most suitable. Different sour­ces of biomass require very different production systems and therefore a variety of sustainability issues can arise. These range from very positive benefits (e. g. use of waste material, or growing woody biomass on degraded agricultural land) through to large-scale diversion of high-input agricultural food crops for bio­fuels (O’Connell et al., 2009).

Contrary to rational engineering, partial and/or addi­tional metabolic pathways of microorganisms can be engineered to enhance bioproduct production. The term "metabolic engineering" was first coined by Bailey and was described as a vast variety of manipulations and experimental procedures to improve the productiv­ity of a desired metabolite by an organism (Bailey, 1991). More specifically, examples of metabolic engineering can include increased productivity and/or yield, improvement of substrate uptake, widening the scope of substrate range for an organism, modification of metabolic flux, and elimination of unnecessary or competing metabolic pathways (Stephanopoulos, 1999).

Metabolic engineering, similar to rational engineer­ing, requires the selection of a good host/microor — ganism as a candidate for the production of biofuels and/or bioproducts from biomass. This could include engineering desired pathways into well-studied host microorganisms such as Escherichia coli and Saccharo — myces cerevisiae; these microorganisms have been used for industrial-scale production for several years. How­ever, some experts suggest that engineering desired pathways into microorganisms that already possess industrial properties may be more successful. This is due to the potential for metabolic burden to the cell; new metabolic pathways require amino acids, redox cofactors, and energy for synthesis and function of its enzymes (Lee et al., 2008a).

Furthermore, metabolic engineering poses several general challenges for researchers including the devel­opment of recombinant DNA technologies for selected host microorganisms, development of quantitative tools, methods to understand flux modification in complex biological systems, and the development of quantitative techniques to determine changes in fluxes or metabolite concentrations (Cameron and Tong, 1993). A few suc­cessful examples of metabolic engineering to improve general host and select host microorganisms metabolism for the digestion and conversion of biomass are outlined below.

Recently, the development of genome-scale modeling permits the prediction of how new metabolic pathways may impact growth and product production using meta­bolic models. These models result in a more rational approach to metabolic engineering (Patil et al., 2004). Moreover, stoichiometric models can be defined by established equations through the use of metabolic flux analysis (MFA); this is established by measuring ex­change fluxes experimentally (Lee et al., 2008b). For example, the native metabolism of E. coli under different growth conditions (Kayser et al., 2005) and during re­combinant protein production (Ozkan et al., 2005) has been determined using MFA. For efficient application in biofuel and bioproduct production, genome-scale models should be developed with constraints to opti­mize flux in desired pathways, while balancing impor­tant cofactors and energy metabolites (Lee et al., 2008b).

Host microorganisms such as E. coli and S. cerevisae have been improved time and again for the fermentation of sugars to ethanol. In particular, due to the broad range of carbohydrates metabolized by E. coli, it has been a po­tential candidate for the expression of ethanologenic pathways in some studies. For example, a portable cassette called the production of ethanol operon (PET operon) was used to genetically engineer the homoetha — nologenic pathway from Zymomonas mobilis into E. coli, which included the pyruvate decarboxylase and alcohol dehydrogenase B genes. Using the PET system, these genes were integrated into the chromosome of E. coli at the pfl locus. Meanwhile the fumarate reductase (frd) gene was deleted to eliminate succinate production, therefore preventing carbon loss. These metabolic changes resulted in the recombinant strain KO11, which produced ethanol yields as high as 95% in complex me­dium (Jarboe et al., 2007; Ohta et al., 1991). However, host strains such as E. coli may encounter metabolic bur­dens and are often not naturally adapted to the toxicity of end products like ethanol. Thus, there have also been some attempts to metabolically engineer known biomass-converting bacteria or fungal strains.

Typically, bacteria produce more desirable end prod­ucts through facultative and anaerobic digestion, as is the case for bacteria belonging to the class Clostridia. Much of the metabolic engineering in these species fo­cuses on product formation, which may include the elimination of undesirable products such as in the case of an engineering project conducted on Clostridium acetobutylicum—a well-known ethanogenic strain stud­ied often for the production of butanol. In brief, the ace- toacetate decarboxylase gene (adc) was disrupted in the hyperbutanol-producing strain C. acetobutylicum EA 2018 using TargeTron technology (Sigma Aldrich) (Jiang et al., 2009). TargeTron is a group II intron developed for rapid and site-specific gene disruption in prokaryotes. The disruption of adc led to an increase in butanol ratio from 70% to 80.05%, with a simultaneous reduction in acetone of 0.21 g/l (Jiang et al., 2009).

In contrast, one can implement metabolic engineering to improve native metabolism in microorganisms by engineering entirely novel pathways for desired product formation, which is more practically done in hosts able to hydrolyze biomass, such as the example with Clos­tridium cellulolyticum. Recently, Higashide et al. demon­strated the production of isobutanol from crystalline cellulose in C. cellulolyticum (Higashide et al., 2011). In this study, the development of valine biosynthesis pathway required the expression of five genes, alsS, ilvC, ilvD, kivD, and ahdA, to convert pyruvate into iso­butanol. Consequently, only the expression and function of kivD (2-keto-acid decarboxylase) and alsS (alpha — acetolactate synthase) were confirmed; nonetheless modified C. cellulolyticum produced up to 660 mg/l of isobutanol over a 7- to 9-day growth period (Higashide et al., 2011).

These examples of engineering and modeling to improve the metabolic capabilities of strains helped lay the foundation for future development of biomass­converting microorganisms. Combined with the ability to rationally design enzymes with greater stability and/or increased specific activity the modification of microorganisms in industrial production of biofuels and bioproducts looks promising.

There are numerous investigations on the mecha­nisms of electron transfer to the anode by the microbes, while the reports about electron transfer to a biocathode are rather limited (Lovley, 2008). The two electron trans­fer directions are opposite. The biocathode is an electron donor while the anode is an electron acceptor. Despite this difference, biocathodes use the same electron trans­fer mechanisms, DET and MET (Rosenbaum et al., 2011), because biofilm electron transfer can be bidirectional.

DET for Biocathodes

Similar to DET for anodes, DET for biocathodes also requires physical contact of the microbial cell wall with the electrode surface. At the site of direct contact, the electrons directly transfer to the outer cell membrane-bound redox macromolecules (such as c-type cytochromes) from the electrode (Figure 9.4(a); Huang et al., 2011c). However, this kind of DET can only utilize a monolayer of sessile cells on the cathode, thus limiting the biocathode performance. With an in­crease in biofilm thickness, the power generation decreased due to mass transfer resistance to oxidant diffusion from the bulk fluid to the cathode surface (Behera et al., 2010). Geobacter species and mixed cultures that use nitrate, fumarate, tetrachloroethene, O2, CO2, U(VI)/U(IV), and so on as an electron acceptor generally transfer the electrons via DET (Table 9.2). On biocathodes, most of the microbes are found to be Gram negative although some Gram-positive microbes exhibit the DET mechanism in cyclic voltammetry (Huang et al., 2011c). Compared with the pure culture

systems, the mixed culture biocathodes also can transfer electrons via DET. When nitrate, carbon dioxide or tri — chloroethene is used as the electron acceptor, the DET in the mixed culture biocathode improves the power generation (Aulenta et al., 2010; Cao et al., 2009; Clau — waert et al., 2007a).

Microalgae are a heterogeneous group of organisms consisting of both prokaryotes such as cyanobacteria and eukaryotes such as diatoms (Bacillariophyta), dinoflagelates (Dinophyta), green algae (Chlorophyta), yellow-green algae (Xanthophyta), and red algae (Rhodophyta) (Brennan and Owende, 2010; Hu et al.,

2008) . Similarly, other oleaginous microorganisms are defined as microorganisms with lipid content in excess of 20%. The number of bacteria that produce lipids that could be used for biodiesel production is very small. As a result, bacteria are mainly used for special lipid produc­tion such as Docosahexaenoic acid (DHA). Many yeasts and fungi also produce high quantities of lipid. Yeasts with high lipid content include Candida curvata (58%), Cryptococcus albidus (65%), Lipomyces strakeyi (64%) and Rhodotorula glutinous (72%). Oleaginous fungi include Aspergillus oryzae (57%), Mortierella isabellina (86%), Humi — cola lanuginose (75%) and Mortierella vinacea (66%) (Meng et al., 2009). In terms of microalgae, species are generally unicellular organisms but there are also a number of sim­ple multicellular organisms that occur as colonial or fila­mentous groups of cells. Microalgae are capable of autotrophic, heterotrophic and mixotrophic growth. Microalgae populate a wide variety of ecological niches due to a wide range of tolerance for various growth con­ditions such as availability of nutrients, salinity, pH and temperature (Brennan and Owende, 2010; Gong and Jiang, 2011; Schenk et al., 2008). Currently, microalgae contribute very little biolipid to the overall bioenergy market as full-scale commercialization has yet to be realized. Despite this fact, microalgae remain the feed­stock with the greatest potential for supplying future demand for bioenergy in the form of liquid fuels. The idea of using microalgae as a source of biolipids for bio­fuel is not a new one, however. For example, the Aquatic Species Program was launched in 1978 by what is now known as the National Renewable Energy Laboratory (NREL) with its main focus being, "the production of bio­diesel from high lipid-content algae grown in ponds, uti­lising waste CO2from coal fired power plants" (Sheehan et al., 1998). Over 3000 microalgae strains were initially collected, 300 of which were eventually identified as oil rich. When the program was officially closed in 1998 the conclusions were that no "fundamental engineering and economic issues" were identified that would hamper the feasibility of large-scale microalgae culture. The au­thors noted, however, that total biomass and algal lipids produced were still below "theoretical potential, and the requirements for economic viability" (Sheehan et al., 1998). The economic viability was, of course, based on a time when oil prices in the United States were among their all-time lowest at less than $20 per barrel (adjusted for inflation). Today the average oil price is approxi­mately $100 per barrel and this, along with increased pressure to reduce GHG emissions as well as significant technical advances, has made microalgae-derived bio­fuels even more relevant to meet current bioenergy demands.

Gasification

Gasification of biomass is to convert it into useful gases such as carbon monoxide, hydrogen and light hydrocarbons (Brown, 2003). Since the mid-1980s, inter­est has grown on the subject of catalysis for biomass gasification. The advances in this area have been driven by the need for producing tar-free gases from biomass. The avoidance of tars and the yield of hydorgen are deciding factors the economic viability of the biomass

gasification process. Major reactions in gasification are as follows (Brown, 2003).

1

C + — O2 4 CO

2 2

C + CO2 4 2CO C + H2O 4 H2 + CO C + 2H2 4 CH4 CO + H2O 4 H2 + CO2 CO + 3H2 4 CH4 + H2O

The desired product from gasification of biomass is hydrogen or syngas. Syngas can be burned directly in gas engines, be used to produce methanol, or be con­verted into synthetic fuels via the Fischer—Tropsch process. Though gases are target products, gasification of biomass leaves behind solid residuals such carbon and inorganic compounds (ash).

Gasification of biomass is normally performed in the presence of steam and the process depends on the occur­rence of the steam-reforming reactions. Water, in the form of steam, is often added to promote additional produc­tion of hydrogen via the water—gas shift reaction. As the biomass is heated, moisture contained in the biomass is converted to steam, which can react with biomass. However, in practice, proper drying of biomass before feeding it into gasification equipment is still needed in view of energy-input.

Small amounts of oxygen can also be added to the gas feed. The heat from exothermic oxidation reactions can then be used by the endothermic steam-reforming reac­tion. In addition, oxygen has a function to delay the cata­lyst deactivation by helping burn off some of the coke formed.

Catalytic Gasification

Table 15.2 lists some typical results from the gasifica­tion of lignocellulosic biomass in the presence of catalysts.

Biomass gasification is inevitably accompanied with tar formation. Nevertheless, tar can be effectively minimized by catalytic cracking. Naturally occurring dolomite (CaMg(CO3)2), for example, has been used as a catalyst for gasification of biomass in a fluid bed reactor to reduce the tar content by transforming it to gases (Delgado and Aznar, 1997). The mineral-based catalyst generally con­tains CaO, MgO, CO2 and trace minerals such as SiO2, Fe2O3 and Al2O3. The tar cracking efficiency over the dolomites depends on their chemical composition. In general, dolomites with the lowest content of CaO and MgO show the lowest tar cracking efficiency. Yu et al. gasified birch on the four types of dolomites (deposites in Zhenjiang, Nanjing, Shanxi, and Anhui, China) and a Swedish dolomite (Sala) (Yu et al., 2009). The result was that Anhui dolomite showed a low catalytic capacity to crack tar at 973 and 1073K due to its lowest content of CaO and MgO among the tested dolomites. An alterna­tive can be naturally occurring particles of olivine, which are a mineral containing magnesium oxide, iron oxide and silica. Regarding their attrition resistance, Olivine is advantageous over dolomite (Devi et al., 2005).

Alkali salts are often added to biomass by dry mixing or wet impregnation and used as catalysts for the elim­ination of tar and upgrading of the product gas (Li et al., 1996; Encinar et al., 1998). But it has considerable difficulty in catalyst recovery and disposal of ash. Carbonates, oxides and hydroxides of alkali metals can effectively cata­lyze the decomposition of tar during catalytic gasification (McKee, 1983). Earlier, for example, Mudge et al. investi­gated the catalytic steam gasification of wood using alkali carbonates and naturally occurring minerals (trona, borax), which were either impregnated or mixed with the biomass (Mudge and Baker, 1985). The order of activity reported was potassium > carbonate > sodium carbonate > trona > borax.

The Ni-based catalysts for biomass gasification in a fluid bed reactor are typically Ni-Al based one (Garcia et al., 2002; Arauzo et al., 1997) and Ni/olivine one (Courson et al., 2002, 2000). Ni catalysts help to remove tars and methane and to adjust the composition of syn­thesis gas. Sinag et al. studied the effect of nano-sized and bulky ZnO and SnO2 at 573 K on the water-gas shift reaction in gasification of cellulose. The results showed that the water-gas shift reaction proceeded faster over ZnO catalysts than that over SnO2 catalysts. Therefore, a higher yield of hydrogen was obtained in the presence of ZnO (Sinag et al., 2011).

However, catalysts often suffer from deactivation by sintering and/or coke deposition. The use of supercriti­cal water can prevent catalyst from deactivation by means of extracting the coke precursor from the catalyst surface (Baiker, 1999). In addition, it can improve solubi­lity of cellulosic materials and thus reduce mass-transfer limitation. It is also worth noting that, in addition to the active component in a catalyst, usually the acidity and basicity of a support is also an influential factor on product distribution and coke formation. Tasaka and coworkers disclosed that steam reforming of tar derived from cellulose gasification was efficiently catalyzed by 12 wt% Co/MgO catalyst at 873 K in a fluidized bed reactor (Tasaka et al., 2007).

Supported Ru, Pt or Pd catalysts also appear prom­ising in the catalytic gasification of lignocellulosic biomass. They were able to overcome the shortcomings of Ni-based catalysts and dolomite catalysts, although they are relatively costly. Usui et al. gasified cellulose in hot-compressed water at 623 K in the presence of a series of supported catalysts such as Zr(OH)4, (CH3COCH=C(O-)CH3)3Fe, ferrocene, Ru3(CO)12,

(CH3COCH=C(O-)CH3)2Co, NiC2O4, NiO, Ni(OH)2, PdI2 and Cu(OH)2. After reaction for 3 h, 5 wt% Pd sup­ported on Al2O3 showed the highest catalytic activity, leading to a 42.3 vol% yield of H2 and a 7.7 vol% yield of CH4 (Usui et al., 2000). Tomishige et al. found that the order of M/CeO2/SiO2 catalyst activity in the cedar wood gasification at 823 K was the following: Rh > Pd > Pt > Ni=Ru (Tomishige et al., 2004). For Rh/ CeO2/M-type (M=SiO2, Al2O3, and ZrO2) catalysts for cellulose gasification in a continuous-feeding fluidized — bed reactor, Asadullah et al. found that Rh/CeO2/SiO2 exhibited the best performance in terms of generating syngas or hydrogen (Asadullah et al., 2001, 2003).

Lignins in their native form are the most abundant renewable aromatic polymers on earth (Kirk and Farrell, 1987). Consequently, lignins present great potential as a source of energy due to their high fuel content (26—28 MJ/ton dry lignin) rivaling the fuel content of some coals (Lora, 2006; Tomani, et al., 2011). Lignins can be combusted to produce "green" electricity, power, fuel, steam, or syngas; all these are forms of energy which are being or will be used in the future to operate industrial plants where lignins are generated as by­products. The lignin by-products are called "technical lignins" or "industrial lignins" and they differ dramati­cally in properties from the native lignins found in plants. Examples of the use of technical lignins as a source of energy to run industrial plants are the pulp
mills deployed worldwide and the emerging lignocellu — lose biorefineries. Energy production is, compared to all other technical lignins applications, the one with the lowest market value, estimated at approximately 10 US$ cents/kg as coal replacement (Holladay et al., 2007). However, energy generation is the lignin applica­tion with the highest demand by volume and currently the one with the lowest technical risk. Almost every ma­jor pulp chemical mill today utilizes lignin as a source of energy. The latter is today’s common industrial practice which will likely be mirrored by future cellulosic biomass biorefineries which will use lignin as the main energy source in combination with other fuels such as raw biomass.

Technical lignins are available in large volumes, pri­marily in kraft mill spent liquors ("black liquors"), and, to a less extent, in the spent liquors of the few remaining sulfite mills ("brown liquors"). According to our conservative estimate, ca. 6—7% of the spent liquor

Bioenergy Research: Advances and Applications http://dx. doi. org/10.1016/B978-0-444-59561-4.00018-8

produced at a kraft pulp mill could be used for lignin extraction without significantly affecting the plant energy balance. This represents a potential average lignin production capacity per plant in the order of 30—75 tons of lignin per kraft pulp plant per day (Domtar, 2013) assuming an average annual pulp pro­duction capacity of ca. 0.5 million tons odw pulp (Table 18.1). On the contrary, in sulfite pulp mills, the majority of the produced spent liquor can be used for

lignosulfonate production given the fact that not many sulfite pulping players burn lignosulfonates for energy generation. In 2004, it was reported that 2% of the lignin available in the pulp and paper industry was commer­cially used comprising about 1,000,000 tons/year ligno — sulfonates from sulfite pulping brown liquors and <100,000 tons/year from kraft spent liquors (Gosselink et al., 2004). Assuming the forecasted annual growth rate of 2.5% (IHS-Chemical, 2012), the current lignosul- fonates production should be ca. 1,200,000 tons/year.

The global annual production of chemical pulps is estimated at 150 million tons/year (Vappula, 2011) with an average odw chemical pulp production per plant of ca. 0.5 million tons (Table 18.1) and a total volume of dis­solved lignin in pulp making of ca. 70 million tons glob­ally (Lora, 2010). Assuming a lignin annual production capacity per chemical pulp mill of ca. 27,000 metric tons kraft lignin per year (Domtar, 2013), it can then be concluded that the estimated annual global potential for kraft lignin production capacity from pulp mills is ca. 6—9 million tons depending on the feedstock species and the plant design which is in good agreement with the estimates reported by other authors (Glasser, 2010; Lora,

2010) . When considering the potential replacement of the main petrochemicals, namely, ethylene, propylene, butadiene and benzene, toluene, and xylene (BTX) iso­mers, which are produced at a rate of about 300 million tons/year with a total value of over $400 billion (Lucin- tel, 2013), by lignin it is relevant to consider the current global potential production capacity of technical lignins. It becomes clear from the analysis performed above that in the best case scenario purified technical lignins pro­duced globally at chemical pulp mills could potentially replace a maximum of ca. 2% of the global volume of main petrochemicals. However, the emerging lignocellu — lose biorefinery industry for production of biofuels and chemicals might completely change this picture. For instance, the US Department of Energy estimates that 1.3 billion tons of biomass is available in the United States alone for biorefining into transportation fuels and chemicals (Perlack et al., 2005). This amount of biomass could make available additionally 225 million tons of lignin which could be utilized for power, trans­portation fuels, products and various combinations of the above (Holladay et al., 2007). Assuming that 20% of this biorefinery lignin (45 million tons) will be converted into BTX and linear hydrocarbons, the result could be ca. 10% replacement of these petrochemicals by lignins produced at American biomass biorefineries alone. In other words, if we consider, in addition to the American biorefineries, a scenario where these biomass biorefining technologies will be deployed globally, we could witness, in the future, a hypothetical situation where a large fraction of petroleum-derived BTX, and perhaps of other petrochemicals too, could be replaced by lignin.

Another abundant source of technical lignins, which is often ignored, is the acid-hydrolysis (AH) lignin ("hy­drolysis lignin") which has been produced at Eastern European wood and agricultural wastes AH plants since the mid-1930s with yields in the range of 350—400 kg lignin/ton odw softwood. The annual production of such hydrolysis lignin in the former Soviet Union reached 1.5 million tons by the end of the 1980s. Howev­er, only 30—40% of the hydrolysis lignin was really uti­lized, whereas the rest was disposed in giant landfills nearby the wood hydrolysis plants, creating as a result serious environmental problems caused primarily by autoignition of these deposits. For example, the current lignin waste stocks in the Irkutsk region (Siberia), where only four plants are located, exceed 20 million tons (Rabinovich, 2010) equivalent to ca. 20 times today’s global commercial technical lignin market. The current annual production of AH lignin in Belarus alone is in the order of 100,000 tons (Podterob et al., 2004). The main application of the hydrolysis lignin is the produc­tion of pellets for energy generation. However, highly specialized applications, such as pharmaceutical entero — sorbents, have been successfully developed and commercialized on the basis of purified hydrolysis lignin. An example of these commercial sorbents is the enterosorbent "Polyphepan" (Podterob et al., 2004).

In addition to the low-value energy lignin applica­tion, a wide diversity of high-value industrial applica­tions have been envisioned or industrially realized or demonstrated including uses as novel materials, poly­meric, oligomeric, and monomeric feedstock. Some of these opportunities, such as the use of lignin or its derivatives in animal feed additives, agriculture, con­struction, textile, oil drilling, binders, dispersants, and composites, are today commercial realities but many others such as the production of carbon fiber precur­sors, the broad incorporation of lignin in synthetic polymeric blends, or the production of BTX remain longer term opportunities with great value and market potential.

Both low — and high-value lignin applications are often seen as efficient vehicles to increase the productiv­ity, reduce fossil fuel consumption, and increase the profitability of the industrial plants where lignin is pro­duced as a by-product. For instance, the Lignoboost™ process (Tomani, 2010), a recently commercialized pro­cess by Metso Corporation (Helsinki, Finland) for lignin production from alkaline black liquors, significantly im­proves the profitability of the pulp and paper mill by debottlenecking the wood pulp production as a result of increasing the recovery capacity of pulping chemicals and valorizing the lignin stream. Commercial-scale lignin production based on the Lignoboost™ process has begun in February 2013 by Domtar Corp. at the Ply­mouth Mill (NC, USA) with a targeted rate of 75 tons / day (~ 27,000 tons/year), destined for a wide range of industrial applications as a bio-based alternative to the use of petroleum and other fossil fuels (Domtar,

2013) .

As it was mentioned earlier, in the case of emerging industries, such as the cellulosic ethanol industry, the smart utilization of residual lignin could dramatically boost the profitability of the cellulosic biofuel plants if converted into value-added chemicals such as BTX, other monomeric and oligomeric phenolic compounds, and suitable for material applications macromolecules such as carbon fiber precursors, polymeric blends, ad­hesives, dispersants, and others. While energy and monomeric applications for technical lignins and their derivatives often target direct replacements of fossil fuels and petrochemicals, the development of novel lignin-derived oligomeric and macromolecular entities has the potential of generating better alternatives or synergy with petrochemical feedstocks. Recent exam­ples of the latter have been reported in the literature which illustrates this concept. For instance, recently Berlin (2011) showed that the replacement of methylene diphenyl diisocyanate (MDI) in engineered wood diiso­cyanate adhesives by organosolv (OS) lignin deriva­tives can lead to substantial improvements of the adhesive binding properties (increased modulus of rupture and modulus of elasticity) when applying the lignin—MDI adhesives in engineered wood composite construction materials such as Oriented Strand Boards (OSB) while still meeting the industry standard require­ments for these adhesives. A similar observation was documented when phenol in phenol—formaldehyde resins was replaced by OS lignin derivatives which resulted in a significant increase of the resin normalized bond strength (Berlin, 2012b). These two examples are important because they illustrate a fact often overseen which is the evidence that lignin derivatives can techni­cally outperform petrochemicals when used in conjunc­tion with the latter in certain chemical formulations. This observation hints at the possibility of not needing to completely depolymerize lignin, a longstanding un­resolved challenging technical problem, into the equiv­alent petrochemical monomers in order to achieve similar or better performance of the lignin-derived chemicals in formulated products. On the contrary, further research efforts could be directed toward valo­rization strategies of technical lignins with preserve natural backbone structures to produce viable novel polymeric precursors alternative to petrochemicals.

The recent resurgence of interest in lignin as a renew­able raw material feedstock is evidenced by the growing number of patent applications containing the word "lignin" which have been filed between 2003 and 2012 via the World Intellectual Property Organization (WIPO; Figure 18.1). It is interesting to note the fact

FIGURE 18.1 Number of WIPO patent applica­tions containing the word "lignin" found in May 2013 by using the WIPO search tool Patentscope for the period 2003-2012.

that 25% of all these patent applications were filed by major chemical, pharmaceutical, and energy companies such as BASF, Bayer, Ciba-Geigy, Monsanto, Sumitomo, and Shell with the German chemical giants Bayer (10% lignin filings) and BASF (8% lignin filings) leading the group. The patents found in May 2013 by using the WIPO search tool Patentscope for the period 2003­2012 (25,974 documents) represent ca. 50% of all the pat­ents registered in the WIPO which contain the word "lignin" on the front page (52,895 documents).

Today’s global lignin market is dominated by the Norwegian company Borregaard Lignotech (Norway) followed by Tembec (North America-France) and MeadWestvaco (USA). There are a number of smaller players such as Domsjo (Finland-India), Granit SA (Switzerland), and CIMV (France), among others.

Lignocellulosic (or second-generation) bioenergy is being intensively developed, due to its use of renewable but nonfood or feed feedstocks; valorization of agricul­tural, forestry, first-generation bioenergy, or municipal by-products or waste; and potential to significantly replace fossil feedstocks for energy or chemical indus­tries. From lignocellulosic biomass materials, cellulose and hemicellulose are converted by (hemi)cellulolytic enzymes or chemical means to fermentable sugars (mainly glucose and xylose), which are then fermented by yeast or bacteria to ethanol or other chemicals. The processes could run alone, fed by selected biomass feed­stocks, or along with the starch/sugar bioethanol or bio­diesel processes, fed by the lignocellulosic by-products from first—generation bioenergy processes.

The main by-product from lignocellulosic bioenergy processes (Figure 20.8) is lignin or lignaceous residue, whose valorizations are the focus of rigorous research efforts and may include uses for the production of phe — nolics (e. g. vanillin, vinyl guaiacol, ferulic acid), lignans, carbon fiber, or as additives for paper and pulp industry, roadbed construction, or soil augmentation (Ceylan et al., 2012; Chapters 22, 23 of this book). Other by­products from lignocellulosic bioenergy processes include stillage and pretreatment liquor, which may be inhibitory to fermentation or enzymatic hydrolysis but rich in phenolics and oligosaccharides (Klinke et al., 2002; Persson et al., 2002). Mechanically separated biomass components (upstream to pretreatment, hydro­lysis, and fermentation) may also serve as sources for
phytochemicals, such as tree barks for tannin production.

Woods, especially those not suited for conventional forestry products, are attractive feedstocks for lignocel — lulosic bioenergy. Prior to enzymatic or chemical conver­sion to fermentable sugars, woody materials might be subjected to treatments (such as the leaching processes widely used for dedicated phytochemical production, as mentioned in Section (Extraction and Isolation from Specific Plants) to yield extractives comprising pheno — lics (phenols, flavonoids, and anthocyanins), terpenoids (essential oils), nitrogen-containing phytochemicals (alkaloids) or organic acids (citric, oxalic, acetic, malic, benzoic, etc.) (Huang and Ramaswamy, 2012; Turley et al., 2006). In addition to agricultural and forestry by-products (e. g. corn stover, wheat straw, and wood residues), switchgrass and other dedicated "energy crops" may serve as viable feedstocks for not only bio­energy but also phytochemical coproducts. For instance, valued phytochemicals like antioxidants and flavonoids might be extracted from switchgrass prior to the pre­treatment of the bioenergy process (Huang and Ramaswamy, 2012; Uppugundla et al., 2009; Wang and Weller, 2006).

Conventional Agricultural Production

Over the decades from 1960 to 2010, agricultural pro­duction has increased more than threefold and per cap­ita provision of calories has increased by one-third (FAOSTAT, 2013). This has greatly contributed to feeding an ever-increasing population (from 3×109 to 6.7×109 billion over this period). This development is usually subsumed under the label "Green revolution" (IFAD, 2001; Evenson and Gollin, 2003). Starting in the 1960s, this development was based on a strict focus on monocropping with high yielding species and varieties, irrigation and mechanization where available and increased use of mineral fertilizers, pesticides and herbi­cides. The successes of the green revolution are evident, but so are the downturns related to it (Matson et al., 1997; DFID, 2004). The focus on monocropping, chemi­cal fertilizers, pesticides, irrigation and mechanization has left an increasingly negative legacy regarding adverse effects on soil fertility, i. e. increased soil degra­dation, salinization and depletion of water bodies, on intoxication of the environment, biodiversity loss, loss of ecosystem services, on eutrophication of water bodies and animal health (Matson et al., 1997). Current agricul­ture is well able to feed the world and will be able in 2050 to feed more than 9 billion people, given projected yield increases realize (Alexandratos and Bruinsma, 2012). The challenge is not the average supply of calories per capita but their distribution globally and the fact that a third is lost or wasted globally (Godfray et al., 2010). However, in the light of the adverse effects of the green revolution and climate change, such yield increases may be compromised and sustained agricultural production calls for alternative cropping practices and a funda­mental shift in the agricultural production system (IAASTD, 2009; Muller et al., 2010).

Sustainable Agricultural Production

A new revolution in agricultural production is thus needed. On the production level, a sustainable future agricultural system needs to focus on mitigating and avoiding the adverse effects of current agricultural prac­tices. It needs to focus on crop diversity, ecosystem ser­vices, soil protection and fertility, nutrient and water use cycling, biocontrol of pests, diseases and weeds and reduced pesticide use. A range of alternative pro­duction approaches are available (Eyhorn et al., 2003; Pretty et al., 2006; Rossi, 2012), such as agroecology — based approaches, focusing on utilization of ecological concepts (Altieri, 1995), or integrated pest management, focusing on reducing pesticide use via managing pest populations in such a way that damages remain low (Bajwa and Kogan, 2002). The role model for these alter­native approaches is organic agriculture with its ban on most pesticides, focus on soil fertility, plant health and closed nutrient cycles, utilization of optimized crop rota­tions and crop diversity, organic fertilizers and ecosystem functions for pest and weed control (FAO, 2002; Eyhorn et al., 2003; IFOAM, 2006). Organic agricul­ture is the role model as it addresses all adverse effects of conventional production, adopts a systemic approach and is well established and tested for decades and embedded in a context of governance, information pro­vision, training and extension institutions that make it the best-developed alternative production system. Organic agriculture performs better than conventional agriculture with respect to most environmental indica­tors on a per-hectare basis (Schader et al., 2012). The biggest drawback is its generally lower yields (Seufert et al., 2012; De Ponti et al., 2012; Badgley et al., 2007). Lower yields predominantly manifest in comparison to high-yielding intensive conventional agriculture. In developing countries, in a context of currently no­optimal conventional production systems, organic yields are on par or even higher for well-managed organic farms. The lower yields can result in a less favor­able per kilogram produce assessment of environmental impacts for some products in organic agriculture (Schader et al., 2012). We emphasize that we do not address socioeconomic aspects of organic production here, such as the need for information and extension ser­vices to train farmers and potential challenges of the conversion from organic to conventional agriculture.

Interestingly, the key principles and practices of organic agriculture become increasingly important in conventional agriculture, mainly due to the increasing need to contribute to climate change mitigation and adaptation but also due to the increasingly important discussion on global biodiversity losses. Optimized crop rotations with deep-rooting forage legumes and use of organic fertilizers, for example, are promoted in the context of climate change mitigation and adaptation to improve soil fertility and increase soil organic carbon levels (Smith et al., 2008) and reducing nitrogen loads are key to protect biodiversity.